Methods and compounds for modulating the secretion or expression of adhesion proteins or angiopoietins of cells

ABSTRACT

This invention provides methods, processes, compounds and compositions for modulating the gene expression or secretion of adhesion proteins, angiopoietins or their receptors to cure diseases, for anti-angiogenesis and for treating parasites, wherein the adhesion proteins or receptors comprise fibronectin, integrins family, myosin, vitronectin, collagen, laminin, glycosylation cell surface proteins, polyglycans, cadherin, heparin, tenascin, CD 54, CAM, elastin and FAK; wherein the angiopoietins comprise angiopoietin 1, angiopoietin 2, angiopoietin 3, angiopoietin 4, angiopoietin 5, angiopoietin 6, angiopoietin 7, angiopoietin-like 1, angiopoietin-like 2, angiopoietin-like 3, angiopoietin-like 4, angiopoietin-like 5, angiopoietin-like 6, and angiopoietin-like 7; wherein the cancers comprise breast cancer, leukocyte cancer, liver cancer, ovarian cancer, bladder cancer, prostate cancer, skin cancer, bone cancer, brain cancer, leukemia cancer, lung cancer, colon cancer, CNS cancer, melanoma cancer, renal cancer, cervical cancer, esophageal cancer, testicular cancer, spleenic cancer, kidney cancer, lymphatic cancer, pancreas cancer, stomach cancer and thyroid cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-part of U.S. Ser. No. 15/181,631,filed Jun. 14, 2016, which is a continuation of U.S. Ser. No.13/841,053, filed Mar. 15, 2013, which is a Continuation-in-part of U.S.Ser. No. 13/718,575, filed Dec. 18, 2012, and International App'l No.PCT/US2009/034115, filed Feb. 13, 2009, which claims benefit of U.S.Ser. No. 61/038,277 filed Mar. 20, 2008, U.S. Ser. No. 61/054,308, filedMay 19, 2008, and is a Continuation-in-part of U.S. Ser. No. 12/344,682,filed Dec. 29, 2008 and International App'l No. PCT/US2008/002086, filedFeb. 15, 2008, which is a continuation-in-part of International App'lNo. PCT/US2007/077273, filed Aug. 30, 2007, which claims benefit of U.S.Ser. No. 60/890,380, filed on Feb. 16, 2007, U.S. Ser. No. 60/947,705,filed on Jul. 3, 2007, and is a continuation-in-part of U.S. Ser. No.11/683,198, filed on Mar. 7, 2007, which claims benefit of U.S. Ser. No.60/795,417, filed on Apr. 27, 2006, 60/841,727, filed on Sep. 1, 2006,60/890,380, filed on Feb. 16, 2007, and is a continuation-in-part ofInternational Application No. PCT/US2006/016158, filed Apr. 27, 2006,which claims benefit of U.S. Ser. No. 60/675,282, filed Apr. 27, 2005and U.S. Ser. No. 60/675,284, filed Apr. 27, 2005 and is acontinuation-in-part of the following applications (1) U.S. Ser. No.11/289,142, filed Nov. 28, 2005; (2) U.S. Ser. No. 11/267,523, filedNov. 4, 2005; (3) International Application No. PCT/US05/31900, filedSep. 7, 2005 (which claims the benefit of U.S. Ser. No. 60/617,379,filed Oct. 8, 2004, 60/613,811, filed Sep. 27, 2004, and 60/607,858,filed Sep. 7, 2004); (4) U.S. Ser. No. 11/131,551, filed May 17, 2005;and (5) U.S. Ser. No. 11/117,760, filed Apr. 27, 2005. This applicationis also a continuation-in-part of U.S. Ser. No. 11/412,659, filed Apr.27, 2006, Ser. No. 10/906,303, filed Feb. 14, 2005, Ser. No. 11/117,745,filed Apr. 27, 2005, Ser. No. 12/195,112, filed Aug. 20, 2008, Ser. No.12/392,795, filed Feb. 25, 2009, Ser. No. 12/541,713, filed Aug. 14,2009, and Ser. No. 12/714,598, filed Mar. 1, 2010. The contents of thesepreceding applications are hereby incorporated in their entireties byreference into this application.

FIELD OF THE INVENTION

This invention identifies Xanifolia-Y's cellular target(s). Xanifolia-Yis an alternate or supplemental anticancer agent to other DNA-inhibitionor microtubule-targeting drugs.

In an embodiment, Xanifolia-Y binds adhesion proteins to blocks themigration, metastasis angiogenesis of cancer cells. It inhibits thegrowth of cancers. The compounds in this application have effects oncell membrane structure and cell's adhesion process. This inventionrelates to the inhibiting cancer through regulating aquaporin in cancercells and/or interacting with aquaporin with compounds comprise of atriterpene with two angeloyl groups. In an embodiment, the compound maybe a saponin wherein comprises at least one angeloyl, preferable twoangeloyl groups. In an embodiment, the compound may comprise more thantwo angeloyl groups, acetyl group, tigloyl group, senecioyl group, or anacid having two to five carbons or combination thereof. This inventionrelates to saponins and compounds with angeloyl groups isolated fromplants, their uses and functions. A composition comprises a diangeloylgroup compound for inhibiting hemorrhoids, venous insufficiency andswelling. The compounds and compositions in this invention inhibit tumoror cancer growth. This invention provides methods and compositions foraffecting the gene expression in cells as a result that cure diseases,wherein the methods comprise reducing the syndrome of diseases. In anembodiment, the method comprises inhibition of gene expression.

In an embodiment the method comprises stimulating the gene expression.This invention provides methods, processes, compounds and compositionsfor modulating the gene expression or secretion of adhesion proteins ortheir receptors to cure disease, wherein the modulating comprisespositive and negative regulating; wherein comprises inhibiting cancergrowth, wherein the adhesion proteins or receptors comprise fibronectin,integrins family, Myosin, vitronectin, collagen, laminin, Glycosylationcell surface proteins, polyglycans, cadherin, heparin, tenascin, CD 54,CAM, elastin and FAK; wherein the cancers comprise cancer of breast,leukocyte, liver, ovarian, bladder, prostate, skin, bone, brain,leukemia, lung, colon, CNS, melanoma, renal, cervix, esophagus, testis,spleen, kidney, lymph, pancreas, stomach and thyroid.

BACKGROUND OF THE INVENTION

We have identified an herbal extract that inhibits cancer cell's growth.The active compounds were purified and their structures identified to benovel triterpenoid saponins. Varicose veins are swollen and knottedveins that can occur in any part of the body, especially in the calf,inside leg or around the anus. Escin has been satisfactorily used fortreating Varicose veins and chronic venous insufficiency for many years.Escin is a mixture of saponins found in the seed of the horse chestnuttree, Aesculus hippocastanum L., Hippocastanaceae. Escin is the majoractive ingredient prepared from Aesculus hippocastanum(Hippocastanaceae), the horse chestnut tree. In one controlled trialstudy, aescin was shown to be as effective as compression therapy as analternative to medical treatment for CVI. The therapeutic benefit iswell supported by a number of experimental investigations in differentanimal models. See Department of Pharmacological Sciences, University ofMilano, Via Balzaretti 9, 20133 Milano, Italy. New saponin compoundswith two angeloyls have been provided in International PCT ApplicationNo. PCT/US04/33359, filed Oct. 8, 2004, and U.S. Ser. No. 10/906,303.Yingjie Chen, Tadahiro Takeda and Yukio Ogihara reported four newsaponin compounds that were isolated from the fruits of Xanthocerassorbifolia Bunge in Chem. Pharm. Bull., 33(1)127-134, 1985;33(3)1043-1048, 1985 and 33(4)1387-1394, 1985. Other related studies onsaponin compounds include: triterpenoid saponins and acylatedprosapogenins from Harpullia austro-caledonica (Voutquenne et al. 2002);six triterpennoid saponins from Maesa laxiflora (Zhong et al. 1999); newtriterpene saponin from Pittosporum viridiflorum from the Madagascarrainforest (Young et al. 2002); anti-HIV-1 protease triterpenoidsaponins from the seeds of Aesculus chinensis (Yang et al. 1999);triterpenoid saponins from the roots of Camellia sinensis var. assamica(Lu et al. 2000); new acylated triterpenoid saponins from Maesalaceceolata (Apers et al. 1999); isolation and structure elucidation offour new triterpenoid estersaponins from fruits of the PittosporumtobiraAIT (D'Acquarica et al. 2002) and method for the prevention andtreatment of chronic venous insufficiency (U.S. Pat. No. 6,210,680). Thecontents of the above-mentioned references are hereby incorporated byreference.

Human cells are surrounded by aquatic environments. Aquaporins is afamily of transmembrane water-channel transporting proteins that play amajor role in trans-cellular and transepithelial water movement. Thisinvention shows that the triterpene saponins with two angeloyls havestronger activity for inhibiting cancer cell growth by affectingmembrane functions. In an embodiment they affect the aquaporin andpermeability of cell membrane.

SUMMARY OF THE INVENTION

In accordance with these and other objects of the invention, a briefsummary of the present invention is presented. Some simplifications andomissions may be made in the following summary, which is intended tohighlight and introduce some aspects of the present invention, but notto limit its scope. Detailed descriptions of a preferred exemplaryembodiment adequate to allow those of ordinary skill in the art to makeand use the invention's concepts will follow in later sections.

This invention provides the uses of compounds comprising a triterpene orother sapongenin with two angeloyl groups, or at least two side groupsselected from the following groups: angeloyl, tigloyl and/or senecioylgroups, wherein the side groups are attached to carbon 21, 22 or/and 28of triterpenoidal saponin, triterpenoid, triterpenoidal compounds orother sapongenin backbones. The methods of purification anddetermination of structures of the compounds are detailed in theInternational Application No. PCT/US05/31900, filed Sep. 7, 2005, andU.S. Ser. No. 11/289,142, filed Nov. 28, 2005. Details also in U.S. Ser.No. 11/131,551, filed May 17, 2005

We have identified an herbal extract from Xanthoceras sorbifolia thatinhibits cancer cell's growth. The active compounds were purified andtheir structures identified to be a novel triterpenoid saponin. We namedthem as Xanifolia-Y and family. Details in U.S. Ser. No. 10/906,303 andInt'l App'l No. PCT/US04/33359

In vivo studies with Xanifolia-Y employing human ovarian carcinomaxenografts in mouse indicate that Xanifolia-Y is capable of extendingthe life span of animals bearing human tumors. These results show thatit can be useful in treating cancers in mammal. In an embodiment it canbe use in treating human cancers, preferably ovarian cancer. Xanifolia-Yprolongs the life span of mice bearing of human tumor. It blocks themigration or metastasis of cancer cells. In an embodiment it binds withadhesion proteins or interferes with the function of molecules oncarcinoma cells or on the mesothelial cells. It inhibits the tumorgrowth in mammal. It is useful in cancer therapy. See Experiment 7, 8,9.

Xanifolia-Y binds with adhesion proteins or signaling proteins in cancercells. Xanifolia-Y is radioactive labeled with ³H and use it as ligandto search for target molecules. With the labeled Xanifolia-Y, we studythe cellular binding location with autoradiography; determine bindingaffinity to adhesion proteins or target protein with RIA, investigateits associated proteins with co-IP and verify them with competitionassay. In an embodiment, Xanifolia-Y binds to adhesion proteinscomprising integrins family, CD44, fibronectin, Myosin VI or FAK. Weidentify the target proteins by 2D gel blotting and MALDI-TOF peptidemapping techniques. Xanifolia-Y is inhibiting nodule formation and/orgrowth in the peritoneal cavity of mouse. Cancer cell is inoculated intothe peritoneal cavity of nude mice. Drug treatment starts at differentstages of tumor progression. At the end of the drug-treatment, thechange in the number and weight of tumor nodules are measured.Xanifolia-Y is inhibiting solid tumor growth. Details are in Experiment10.

Xanifolia-Y has effects on cell membrane structure and adhesion process.This invention identifies Xanifolia-Y's cellular target(s). It is analternate or supplemental anticancer agent to other DNA-inhibition ormicrotubule-targeting drugs. In an embodiment, this invention provides amethod of binding with adhesion proteins to blocks the migration,metastasis of cancer cells, anti-angiogenesis and inhibits the growth ofcancers.

This invention discloses saponin compounds having the specificstructures are capable of inhibiting cancer or tumor cell growth andanti-angiogenesis. This invention provides a method for treating cancerwherein the cancers comprise breast cancer, leukocyte cancer, livercancer, ovarian cancer, bladder cancer, prostate cancer, skin cancer,bone cancer, brain cancer, leukemia cancer, lung cancer, colon cancer,CNS cancer, melanoma cancer, renal cancer or cervix cancer. Thisinvention provides a method for treating cancer by binding with adhesionproteins to blocks the migration, metastasis of cancer cells, growth ofcancers wherein the cancers comprise breast cancer, leukocyte cancer,liver cancer, ovarian cancer, bladder cancer, prostate cancer, skincancer, bone cancer, brain cancer, leukemia cancer, lung cancer, coloncancer, CNS cancer, melanoma cancer, renal cancer or cervix cancer. Thisinvention relates to the mechanism of inhibiting cancer by regulatingaquaporin in cancer cell and the interacting of aquaporin with compoundscomprise a triterpene and two angeloyl groups. In an embodiment, thecompound may be a saponin wherein comprises at least one angeloyl,preferable two angeloyl groups.

This invention relates to the aquaporin pathway that is influenced bysaponins with angeloyl groups in inhibiting cancer. This inventionrelates to a method for curing enuresis, frequent micturition, urinaryincontinence by regulating the aquaporin with a compound whereincomprises a triterpene, angeloyl group(s) and sugar moiety. Varicoseveins are enlarged veins that can be flesh colored, dark purple or blue.They often look like cords and appear twisted and bulging. They areswollen and raised above the surface of the skin. Varicose veins arecommonly found on the backs of the calves or on the inside of the leg.During pregnancy, varicose veins called hemorrhoids can form in thevagina or around the anus. This invention provides the uses ofcompositions for treating or preventing chronic venous insufficiency,peripheral edema, antilipemic, chronic venous disease, varicose veindisease, varicose syndrome, venous stasis, expectorant, peripheralvascular disorders, cerebro-organic convulsion, cerebral circulationdisorder, cerebral edema, psychoses, dysmenorrheal, hemorrhoids,episiotomies, peripheral edema formation or postoperative swelling; forreducing symptoms of pain; for reducing symptoms of stomach pain; forreducing symptoms of leg pain; for treating pruritis, lower leg volume,thrombosis, thromophlebitis and for treating or preventing gastriculcers or use for antispasmotic. This invention also provides acomposition for inhibiting tumor cell growth. This invention furtherprovides a composition for preventing tumor formation or killing tumorcells. This invention further comprises the composition of an effectiveamount of compound used for manufacture of a medicament for thetreatment of varicose vein disease, chronic venous insufficiency,hemorrhoids or inhibition of leg swelling.

This invention provides methods and compositions for modulating thesecretion, expression, or synthesis of adhesion protein or angiopoietinof cancer cell and block their migration, metastasis or inhibit thegrowth of cancers or anti-angiogenesis, wherein the adhesion protein andtheir receptors comprise fibronectin, integrins family, Myosin,vitronectin, collagen, laminin, Glycosylation cell surface proteins,polyglycans, cadherin, heparin, tenascin, CD 54, CAM, elastin and FAK.

This invention provides a method of reducing the adhesion protein incell and blocks the migration, metastasis of cancer cells or inhibitsthe growth of cancers or anti-angiogenesis, wherein the adhesionproteins or its receptors comprise fibronectin, integrins family,Myosin, vitronectin, collagen, laminin, Glycosylation cell surfaceproteins, polyglycans, cadherin, heparin, tenascin, CD 54, CAM, elastinand FAK. In an embodiment, this invention provides a method of reducingthe secretion of fibronectin. In an embodiment, this invention providesa method for inhibiting the expression of adhesion proteins, wherein theadhesion proteins comprise fibronectin, integrins family, Myosin,vitronectin, collagen, laminin, Glycosylation cell surface proteins,polyglycans, cadherin, heparin, tenascin, CD 54, CAM, elastin and FAK.This invention provides a method of inhibiting the growth, migration,metastasis of cancer by altering the characteristics of membrane ofcancer cells, wherein the characteristics comprise adhesion protein;wherein the cancers comprise breast, leukocyte, liver, ovarian, bladder,prostate, skin, bone, brain, leukemia, lung, colon, CNS, melanoma, renaland cervix cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. A and B are Structures of saponins.

R1=angeloyl, tigloyl, senecioyl, H, or a sugar moiety comprisingangeloyls, tigloyl or senecioyl. R2=angeloyl, tigloyl, senecioyl, H, ora sugar moiety comprising angeloyls, tigloyl or senecioyl. R6=angeloyl,tigloyl, senecioyl, acetyl, H, or a sugar moiety comprising angeloyls,tigloyl or snecioyl; R3=H or OH. R10=CH₃, CH₂OH, or CHO; R5=D-glucose,D-galactose, L-rhamnose, L-arabinose, D-xylose, alduronic acid,D-glucuronic acid, D-galacturonic acid or H. R7=D-glucose, D-galactose,L-rhamnose, L-arabinose, D-xylose, alduronic acid, D-glucuronic acid,D-galacturonic acid or H; R8=D-glucose, D-galactose, L-rhamnose,L-arabinose, D-xylose, alduronic acid, D-glucuronic acid, D-galacturonicacid or H. R9=COOH or CH₂OH

FIG. 2. Structures of saponins.

FIG. 3. Panels A and B compare potency of compound Y (saponin with 2angeloyl groups) and compound X (saponin with 1 angeloyl) in inhibitinggrowth of ovarian cancer cells. The IC50 for Compound Y is about 1.5μg/ml while 30 μg/ml for compound X. Panel C shows the inhibition ofgrowth of skin cancer cells by the purified compound Y. The IC50 is 0.23μg/ml. Panel D shows the hemolytic activity of Xanifolia-Y, B-Escin,Xanifolia-X, ACH-Y and AKOH-Y

FIG. 4. Panel A: the anticancer activities of Y, Y8, Y9 and Y10,determined by MTT assay on ovarian cancer cells. Panel B: The purifiedcompound Y1 and compound Y2 show inhibition of growth of ovarian cancercells. Panel C: Compound Y inhibits tumor growth (IC50=4 μg/ml).Compound X which has a similar structure to Y but with only one angeloylgroup at C22, has less anticancer activity (IC50=6 μg/ml). Removal ofsugars from Y (ACH-Y) but retaining the diangeloyl group retains 40% ofthe anticancer activity (IC50=9.5 μg/ml). However, removal of bothangeloyl groups from Y (AKOH-Y) completely abolishes its anticanceractivity (even at 120 μg/ml). The results indicate that diangeloylgroups in compound Ys are important for anti-tumor activity. Panel D:Compares inhibition activity of Xanifolia-Y, B-Escin, Xanifolia-X andAKOH-Y

FIG. 5. Comparison of MTT and hemolytic activities of saponin compoundand compound Ys. Panels A and B: Hemolytic activities. Panels C and D:MTT activities.

FIG. 6. Saponin compound Y, X, ACH-Y, AKOH-Y and B-Escin. Thesecompounds are purified and their structures were verified by NMR and MS.These compounds are then used for MTT test.

FIGS. 7-14 show Xanifolia Y1 inhibits Leukemia cancer, Lung cancer,Colon cancer, CNS cancer, Melanoma Ovarian cancer, Renal cancer,Prostate cancer and Breast cancer activities

FIG. 15-22 show Xanifolia Y2 inhibits Leukemia cancer, Lung cancer,Colon cancer, CNS cancer, Melanoma Ovarian cancer, Renal cancer,Prostate cancer and Breast cancer activities

FIG. 23. Animal study result shows Group A Mice—Implanted tumor and nodrug, died on day 19-22; Group B Mice—Implanted tumor and with drug,survived over 50 days; Group C Mice—No tumor and with drug, survivedover 50 days.

FIG. 24. Animal study result shows Group A Mice implanted with tumor andno drug, all died within 24 days; Group D Mice implanted with tumor andwere given drug 9 times from 4th day, all survived; Group E Miceimplanted with tumor and were given drug 10 times from 10th day, halfthe number of mice survived.

FIG. 25. Animal study shows that the tumor size is 45% smaller in micewith drug than the mice with no drug in 10 days period.

FIG. 26. Study apoptosis induced by Xanifolia-Y that apoptosis is amajor form of cell death induced by Xanifolia-Y.

FIG. 27. EM study the effect of Xanifolia on membrane show that patchesof pits were found in the membrane of Xanifolia-Y treated cells (PanelB) but not in cells treated with the DMSO (Panel A) or AKOH-Y (Panel C)controls. These pits have the size from 80 A to 500 A (in diameter). Thepits represent holes formed in the membrane. The pits are arranged in acharacteristic pattern with smaller pits (80 A in diameter) located inthe periphery and the bigger ones (500 A in diameter) in the center. Thebigger holes are resulted from fusing of the smaller holes (Panel D).Membrane image of cells treated with A: DMSO solvent control, 60 min(magnification: ×60,000); B: Xanifolia-Y 5 uM, 60 min. (×60000); C:AKOH-Y, 20 uM, 60 min. (×60000); D: Xanifolia-Y 5 uM, 60 min. (×20000).

FIG. 28. Inhibition effect of Xanifolia and Paclitaxel on cancer cell

FIG. 29. Activities of Ys.

FIG. 30. Animal survival experiment.

FIG. 31. Determination of Aquaporin.

FIG. 32. Compare the potency of Xanifolia Y in ovary and cervix cell.

FIG. 33-35 show Xanifolia Y0 inhibits Leukemia cancer, Lung cancer,Colon cancer, CNS cancer, Melanoma Ovarian cancer, Renal cancer,Prostate cancer and Breast cancer activities.

FIG. 36-38 show Xanifolia Y9 inhibits Leukemia cancer, Lung cancer,Colon cancer, CNS cancer, Melanoma Ovarian cancer, Renal cancer,Prostate cancer and Breast cancer activities.

FIG. 39. Time studies of inhibition of Fibronectin secretion from cancercells (ES2) after incubation of Xanifolia-Y. Fibronectin released inculture medium was determined by Western blot A: (results of experimentF1) Y is Xanifolia compound Y; B: (results of experiment F3); C:(results of experiment F4).

FIG. 40. Inhibition of Fibronectin Secretion by Xanifolia-Y (WesternBlot).

A: result of experiment F5; B: result of experiment F7; C: result ofexperiment F8; D: result of experiment F11; E: result of experiment F12;F: result of experiment F13; G: result of experiment F14B; H: result ofexperiment 14C.

FIG. 41. Inhibition of Fibronectin Secretion by Xanifolia-Y (WesternBlot).

A: result of experiment F23; B: result of experiment F24; C: result ofexperiment F26; D: result of experiment F27; E: result of experimentF29; F: result of experiment F28.

FIG. 42. Inhibition of Fibronectin Secretion by Xanifolia-Y (WesternBlot).

A: result of experiment F30; B: result of experiment F31; C: result ofexperiment F32; D: result of experiment F33A; E: result of experimentF20.

FIG. 43. Increase synthesis of Angiopoietin-2 in ES2 cells byXanifolia-Y treatment.

FIG. 44. Analysis of genesis of blood vessel in xenograft tumor treatedwith compound Y. Panels A and B show the tumor sections taken from micewithout Xanifolia Y treatment. Panels C and D show the tumor sectionstaken from mice with Xanifolia Y treatment. More blood vessels wereobserved in the control Group 1 than those in the drug-treated Group 2

FIG. 45. Experiment shows that Y10 is cytotoxic to Leishmania Major(promastigotes) with IC50 approximately equal to 15 ug/ml.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides the results of a program for screening thebioactive compounds from natural plants. The majority of the plants arefrom the Sapindaceae family, which has 140-150 genera with 1400-2000species. The program is based on our purification methods and biologicalassays including the MTT assay See International Application No.PCT/US05/31900, filed Sep. 7, 2005 and U.S. Ser. No. 11/289,142, filedNov. 28, 2005. Details also in U.S. Ser. No. 11/131,551, filed May 17,2005.

The invention provides compositions comprising triterpenoidal saponinsmay be isolated from plants in the following genus: Acer, Aesculus,Alectryon, Allophylus, Allosanthus, Amesiodendron, Aphania, Aporrhiza,Arfeuillea, Arytera, Atalaya, Athyana, Averrhoidium, Blighia,Boniodendron, Camellia, Camptolepis, Cardiospermum, Castanospora,Chonopetalum, Chouxia, Chytranthus, Conchopetalum, Cossinia, Cubilia,Cupania, Cupaniopsis, Deinbollia, Delavaya, Diatenopteryx, Dictyoneura,Dilodendron, Dimocarpus, Diploglottis, Diplokelepa, Diplopeltis,Dipteronia, Distichostemon, Dodonaea, Doratoxylon, Elattostachys,Eriocoelum, Erioglossum, Erythrophysa, Euchorium, Euphorianthus,Eurycorymbus, Exothea, Filiciurn, Ganophyllum, Glenniea, Gloeocarpus,Gongrodiscus, Gongrospermum, Guindilia, Guioa, Handeliodendron,Haplocoelum, Harpullia, Hippobromus, Hornea, Houssayanthus, Hypelate,Hypseloderma, Jagera, Koelreuteria, Laccodiscus, Lecaniodiscus,Lepiderema, Lepidopetalurn, Lepisanthes, Litchi, Llagunoa, Lophostigma,Loxodiscus, Lychnodiscus, Macphersonia, Maesa, Magonia, Majidea,Matayba, Melicoccus, Mischocarpus, Molinaea, Negundo, Neotina,Nephelium, Otonephelium, Otophora, Pappea, Paranephelium, Paullinia,Pavieasia, Pentascyphus, Phyllotrichum, Pittosporum, Placodiscus,Plagioscyphus, Podonephelium, Pometia, Porocystis, Pseudima,Pseudopancovia, Pseudopteris, Ptelea, Radlkofera, Rhysotoechia,Sapindus, Sarcopteryx, Sarcotoechia, Scyphonychium, Serjania,Sisyrolepis, Smelophyllum, Stadmania, Stocksia, Storthocalyx, Synima,Talisia, Thinouia, Thouinia, Thouinidium, Tina, Tinopsis, Toechima,Toulicia, Trigonachras, Tripterodendron, Tristira, Tristiropsis,Tsingya, Ungnadia, Urvillea, Vouarana, Xanthoceras, Xeropspermum, Zanha,Zollingeria.

This invention provides the uses of compositions comprising atriterpenoidal saponin. In an embodiment, the saponin has triterpenoid,triterpenoidal or other sapongenin, one or more sugar moieties and twoangeloyl groups, or at least two side groups selected from the followinggroups: angeloyl groups, tigloyl groups or senecioyl groups, wherein theside groups are attached to the sapongenin backbone at carbon 21 and 22.In an embodiment, at least two of angeloyl, acetyl, tigloyl, senecioyl,alkyl, benzoyl, dibenzoyl, alkanoyl, alkenoyl, benzoyl alkyl substitutedalkanoyl, aryl, acyl, heterocylic or heteroraryl attached to the sidegroups; wherein the sugar moiety in the saponin comprises at least oneor more of the following sugars and alduronis acids: glucose, galactose,rhamnose, arabinose, xylose, fucose, allose, altrose, gulose, idose,lyxose, mannose, psicose, ribose, sorbose, tagatose, talose, fructose,glucuronic acid, galacturonic acid; or their derivatives thereof, or thecombination thereof; wherein the sugar preferably comprises glucuronicacid, arabinose and galactose.

This invention further provides a composition comprising the structurescomprising at least two side groups selected from the following groups:angeloyl, tigloyl or senecioyl groups, wherein the side groups areattached to a triterpenoidal, triterpenoid, triterpenoidal or othersapongenin backbone. These compositions are obtainable from theabove-identified plants or synthesis. This invention provides a methodof preparing the saponins, comprising the steps of: (a) Extractingroots, kernels, leaves, bark, stem, husks, seeds, seed shells or fruitsof the above plant, or combinations thereof with organic solvents suchas ethanol or methanol to obtain an organic extract; (b) Collecting theorganic extracts; (c) Refluxing the organic extract to obtain a secondextract; (d) Removing the organic solvent from the second extract toobtain a third extract; (e) Drying and sterilizing the third extract toobtain a crude extract powder; (f) Fractionating the crude extractpowder into fractions or components. Fractionation may be achieved byHPLC and FPLC chromatography with silica gel, C18 or other equivalentsolid phase materials; (g) Monitoring the fractionating, if using HPLCor FPLC, the absorption wavelength at 207 nm to 500 nm may be used; (h)Identifying the bioactive components of the crude extract; (i) Purifyingone or more bioactive components of the crude extract with FPLC toobtain one or more fractions of the bioactive component; and (j)isolating the bioactive components with chromatographic techniques thatemploy preparative columns and HPLC. In an embodiment, this inventionprovides the method of MTT Assay to test the bioactivities of thesaponins or other compounds.

Cells.

Human cancer cell lines were obtained from American Type CultureCollection: HTB-9 (bladder), HeLa-S3 (cervix), DU145 (prostate), H460(lung), MCF-7 (breast), K562 (leukocytes), HCT116 (colon), HepG2(liver), U2OS (bone), T98G (brain), SK-MEL-5 (Skin) and OVCAR-3 (ovary).The cells were grown in following culture media: HeLa-S3, DU145, MCF-7,Hep-G2 and T98G are in MEN (Earle's salts); HTB-9, H460, K562 andOVCAR-3 in RPMI-1640; HCT-116 and U20S in McCoy-5A. They aresupplemented with 10% fetal calf serum, glutamine and antibiotics, andincubated in an incubator with 5% CO₂ humidified at 37° C.

MTT Assay.

The procedure for MTT assay followed the method described by Carmichaelet al. (1987) with modifications. The cells were seeded into a 96-wellplate at concentration of 10,000/well for HTB-9, HeLa, H460, HCT116,T98G and OVCAR-3), 15,000/well for DU145, MCF-7, HepG2 and U2OS), and40,000/well for K562 for 24 hours before drug-treatment. The cells werethen exposed to the drugs for 48 hours (72 hours for HepG2 and U2OS, and96 hours for MCF-7). After the drug-treatment, MTT (0.5 mg/mL) was addedto cultures and incubated for an hour. The formazan (product of thereduction of tetrazolium by viable cells) formed and was dissolved withDMSO and the O.D. at 490 nm, and was measured by an ELISA reader. TheMTT level of the cells before drug-treatment was also measured (T0). The% cell-growth (% G) is calculated as: % G=(TD−T0/TC−T0)×100(1), where TCor TD represents O.D. readings of control or drug-treated cells. WhenT0>TD, then the cytotoxicity (LC) expressed as % of the control iscalculated as: % LC=(TD−T0/T0)×100(2).

This invention provides a composition that effectively reduced orinhibitied the cancer cell growth, wherein the cancer includes but isnot limited to bladder cancer, bone cancer and ovary cancer. Thisinvention provides a composition comprising an effective amount oftriterpenoidal saponins named as Xanifolia Y1, Y2, Y, Y7, Y8, Y9, Y10,Y0, their salts or their derivatives for treating chronic venousinsufficiency, peripheral edema, antilipemic, chronic venous disease,varicose vein disease, varicose syndrome, venous stasis, expectorant,peripheral vascular disorders, cerebro-organic convulsion, cerebralcirculation disorder, cerebral edema, psychoses, dysmenorrheal,hemorrhoids, episiotomies, peripheral edema formation or postoperativeswelling; for reducing symptoms of pain; for reducing symptoms ofstomach pain; for reducing symptoms of leg pain; for treating pruritis,lower leg volume, thrombosis, thromophlebitis; for treating rheumatism;for preventing gastric ulcers antispasmotic and inhibiting tumor growth.

This invention provides a method of inhibiting cancer cell growth byaffecting the aquaporin protein. This invention provides a method ofinhibiting tumor growth in a subject comprising administering aneffective amount of compounds in this invention to the subject affectingor interacting the aquaporin protein at the surface of cancer cell. Thecompound comprises two angeloyl groups. In an embodiment the compoundmay be selected from formula (1), (1A), (1B), (1C) and (1D). In anembodiment, the compound comprises a triterpene backbone, two angeloylgroups and sugar moiety. In an embodiment the compound(s) are selectedfrom Xanifolia (Y0, Y1, Y2, Y, Y7, Y8, Y9, and Y10). In an embodimentthe compound(s) are selected from Xanifolia (x), Escin or Aescin. In anembodiment the compound(s) are selected from Compound A to X and A1 toX1 in the application.

This invention provides a method of inhibiting cancer cell growth byincreasing the static charge of the cell, wherein increase water flow inthe cell. In an embodiment the compounsopen the channel protein or iongates of the cells. The charged molecules or ions pass cell membranethrough channel protein and kill cancer cell.

As used herein, the term “inhibit” encompasses prevent, and killing ofthe said cancer or tumor cell

This invention provides a method interacting with aquaporin protein forregulating the water channel, modulating the secretion, regulating thewater metabolism of body, reducing the amount of urine, reducing urinatetimes, treating enuresis, treating frequent urination. The methodcomprises administering an effective amount of compounds to the subjectaffecting or interacting with the aquaporin protein at the surface ofcancer cell. The compound comprises two angeloyl groups. In anembodiment the compound may be selected from formula (1), (1A), (1B) and(1D). In an embodiment, the compound comprises a triterpene backbone,two angeloyl groups and sugar moiety. In an embodiment, the compoundcomprises a triterpene backbone, two acetyl groups with 2 or more carbonand sugar moiety. In an embodiment the compound(s) are selected fromXanifolia (Y0, Y1, Y2, Y, Y7, Y8, Y9, and Y10). In an embodiment thecompound(s) are selected from Xanifolia (x), Escin or Aescin. In anembodiment the compound(s) are selected from Compound A to X and A1 toX1 in the application.

This invention provides uses compound comprises a triterpene andangeloyl groups interacting with aquaporin protein for regulating thewater channel, modulating the secretion, treating enuresis, inhibitingtumor growth, stopping cancer cell proliferate.

In an embodiment, compound interacting with aquaporin protein forregulating the water channel, modulating the secretion, destroying thecancer cell. This invention provides a composition interacting withaquaporin protein for regulating the water channel, modulating thesecretion, treating enuresis, inhibiting tumor growth. A compositioncomprising an effective amount of the compound of any one of Y0, Y1, Y2,Y, Y7, Y8, Y9, Y10, or a salt, ester, metabolite or derivative thereofas a medicament for inhibiting tumor or cancer cell growth and fortreating cancer, wherein the cancers comprise breast cancer, leukocytecancer, liver cancer, ovarian cancer, bladder cancer, prostate cancer,skin cancer, bone cancer, brain cancer, leukemia cancer, lung cancer,colon cancer, CNS cancer, melanoma cancer, renal cancer or cervixcancer.

The aquaporins (AQPs) are a family of homologous water channelsexpressed in many epithelial and endothelial cell types involved influid transport. The family of mammalian AQPs consists of 11 members,AQP0-10, each with a distinct tissue. Functional measurements indicatethat mammalian AQPs 1, 2, 4, 5, and 8 are probably water selective,whereas AQPs 3, 7, 9, and 10 also transport glycerol and other smallsolutes. They are expressed at part of membrane of cell. AQP1 protein isstrongly expressed in most microvessel endothelia outside of the brain,as well as in endothelial cells in cornea, intestinal lacteals, and inother tissues. AQP1 protein was strongly expressed in the membrane ofmicrovessels and small vessels in all ovarian epithelial tumors, butless at the cytoplasm of tumor cells. In addition, AQP1 protein was alsoobserved in the membrane of interstitial cells of ovarian carcinoma.Incorporated by reference of: The influence of aquaporin-1 andmicrovessel density on ovarian carcinogenesis and ascites formation, J.H. Yang et al., 2006 IGCS, International Journal of Gynecological Cancer16 (suppl. 1).

Structural determinants of water permeation through aquaporin-1, byKazuyoshi Murata, et al., Nature, Vol. 407, Oct. 5, 2000. Thedistribution amount of AQPs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 are varied atmembrane of different cells. In different tumor cell, certain type ofAquaporin proteins is over-expressed. An increasing number ofdisturbances have been found associated to abnormal function of theseproteins. The compounds Xanifolia can interact with the aquaporin andinhibit the tumor cell growth. We can use Western blot analysis andidentified expression of aquaporin in cell lines. Detail of Xanifolia inPCT/US05/31900, filed Sep. 7, 2005.

A Western blot is a common method in molecularbiology/biochemistry/immunogenetics to detect protein in a given sampleof tissue homogenate or extract. It uses gel electrophoresis to separateproteins by mass. The proteins are then transferred out of the gel andonto a membrane, where they are “probed” using antibodies specific tothe aquaporin protein. As a result, we can examine the amount ofaquaporin protein in a given sample and compare levels between severalgroups. Other techniques which allow detection of proteins in tissues(immunohistochemistry) and cells (immunoctochemistry) are used. Othermethods such as Bradford protein assay, UV spectroscopy, Biuret proteinassay, Lowry protein assay, Bicinchonic acid protein assay may also beused. There are many publications about the studies of the aquaporin asa maker for cancer cells but none of them mention the regulating oraffecting the aquaporin as method to facilitate the blockage, inhibitionor destroying the cancer cells.

This invention describes a method of destroying cancer cell orinhibiting the cancer cell proliferates by regulating or affecting theaquaporin. In an embodiment, the saponin with two angeloyl groups caninteracts with the aquaporin in order inhibiting the cancer cell growth.In an embodiment the compound may be selected from formula (1), (1A),(1B) (1C) and (1D). In an embodiment, the compound comprises atriterpene backbone, two angeloyl groups and sugar moiety. In anembodiment, the compound comprises a triterpene backbone, two acetylgroups with 2 or more carbon and sugar moiety. In an embodiment thecompound(s) are selected from Xanifolia (Y0, Y1, Y2, Y, Y7, Y8, Y9, andY10). In an embodiment the compound(s) are selected from Xanifolia (x),Escin or Aescin. In an embodiment the compound(s) are selected fromCompound A to X and A1 to X1 in the application. In embodiment themethod is blocking the cancer cell proliferates by regulating,interacting or affecting the aquaporin. In embodiment, the methodincreases the water permeability of the cell membrane by regulating oraffecting the aquaporin in order to kill the cell. In an embodiment, themethod is affecting the aquaporin permitting extra water into the cellto damage the cancer cell. In an embodiment, the method is affecting theaquaporin permitting Glycerol related solute into the cell to damage thecancer cell. In an embodiment, the method is affecting the aquaporinregulating water into the cell to damage the cancer cell, wherein themethod comprise compound selecting from Xanifolia (Y0, Y1, Y2, Y, Y7,Y8, Y9, and Y10). In embodiment, the method is adjusting the fluiddensity outside the cell in order to damage the cancer cell byregulating the fluid pass in the cell wherein the aquaporin isoverexpressed. In an embodiment this invention makes use of the changeof cell membrane permeability to damage the cancer cells. The cellmembrane permeability can be changed by the overexpression of someaquaporins.

The potency of Xanifolia(Y) is difference in different cancer cells ofovary, cervix, lung, skin and breast because they have variation ofAquaporin (AQPs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) at the membrane. Amountof Aquarpoin (AQP 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) at the membrane invarious types of ovarian cancer cells such as OVCAR3, SKOV3, TOV21G andES2 is different, therefore they show different inhibition activity whentreated with Xanifolia (Y). Xanifolia (Y) is a saponin comprises atriterpene, two angeloyl groups and sugar moiety. Xanifolia (Y) inhibitstumor growth (IC50=1.5-4.5 ug/ml). Xanifolia (X) which has a similarstructure to Y but with only one angeloyl group at C22, has lessanticancer activity (IC50=6 ug/ml). Removal of sugars from Y (ACH-Y) butretaining the diangeloyl group retains 40% of the anticancer activity(IC50=9.5 ug/ml). However, removal of both angeloyl groups from Y(AKOH-Y) completely abolishes its anticancer activity (even at 120ug/ml). The results indicate that diangeloyl groups in compound Ys areimportant for anti-tumor activity. The diangeloyl groups pay animportant role in interacting with the aquaporin for inhibiting cancergrowth. The hemolytic activities of human red blood cells by Xanifolia-Y(#63Y), Escin and SIGMA saponin standard were compared. Y contains twoangeloyl groups, Escin has one angeloyl group and SIGMA saponin standardis a mixture of saponins from Quillaia bark. The results show that #63Y(compound Y) has higher hemolytic activity (IC50=1 ug/ml) than Escin orSIGMA saponin standard (IC50=5 ug/ml). See applicationPCT/US2006/016158, filed Apr. 27, 2006, FIG. 6 A. In embodiment, thecompounds of this invention interact with cancer cells and increase thestatic charge of the cells. The static charge increases water flow intothe cells. The cancer cells are collapsed.

This invention describes a method interacting or regulating the proteinon the surface of a cell or altering the functional properties ofintracellular membranes or regulating the fluid passage through the cellwall or softening the skin or improving the skin structure, comprisingadministering to a subject. This invention describes a method ofregulating or affecting the aquaporin, wherein the method comprising theuses of compositions comprising a triterpenoidal saponin. In anembodiment, the saponin has triterpenoid, triterpenoidal or othersapongenin, one or more sugar moieties and two angeloyl groups, or atleast two side groups selected from the following groups: angeloylgroups, tigloyl groups or senecioyl groups, wherein the side groups areattached to the sapongenin backbone at carbon 21 and 22. Wherein thesugar moiety comprises at least one or more of the following sugars andalduronis acids: glucose, galactose, rhamnose, arabinose, xylose,fucose, allose, altrose, gulose, idose, lyxose, mannose, psicose,ribose, sorbose, tagatose, talose, fructose, glucuronic acid,galacturonic acid; or their derivatives thereof, or the combinationthereof; wherein the sugar comprises glucuronic acid, arabinose andgalactose.

The compounds provided in the invention can be used for accelerating thegrowth of bladder, suppressing deep sleep, increasing alertness in asleeping subject, modulating the release, breakdown and uptake ofantidieuretic hormone (ADH) and its receptors; modulating the secretion,breakdown and uptake of adrenocorticotropic hormone (ACTH) and itsreceptors, modulating the release, breakdown and uptake of5-hydroxytryptamine, acetylcholine (Ach), adrenaline (AD), dopamine(DA), norepinephrine (NE) and their receptors; for preventing sleepparalysis, for modulating the formation, release, breakdown and activityof neuropeptides and their receptors. This invention provides a methodcomprising compounds in this invention modulating the secretion,breakdown or uptake of adrenocorticotropic hormone (ACTH) or itsreceptors. This invention provides a composition regulating the proteinon the surface of the cell or alters the functional properties ofintracellular membranes. The compounds and compositions provided in thisinvention can regulate the water passing through the cell wall to softenthe skin or improve the skin structure.

This invention provides a composition comprising the compounds providedin the invention for treating cancers; for inhibiting virus; forpreventing cerebral aging; for improving memory; improving cerebralfunctions, for curing enuresis, frequent micturition, urinaryincontinence, dementia, Alzheimer's disease, autism, brain trauma,Parkinson's disease or other diseases caused by cerebral dysfunctions;for treating arthritis, rheumatism, poor circulation, arteriosclerosis,Raynaud's syndrome, angina pectoris, cardiac disorder, coronary heartdisease, headache, dizziness, kidney disorder; cerebrovascular diseasea;inhibiting NF-Kappa B activation; for treating brain edema, sever acuterespiratory syndrome, respiratory viral diseases, chronic venousinsufficiency, hypertension, chronic venous disease, anti-oedematous,anti inflammatory, hemonhoids, peripheral edema formation, varicose veindisease, flu, post traumatic edema and postoperative swelling; forinhibiting blood clot, for inhibiting ethanol absorption; for loweringblood sugar; for regulating the adrenocorticotropin and corticosteronelevel; and for treating impotence or premature ejaculation or diabetes(See PCT/US05/31900, filed Sep. 7, 2006; U.S. Ser. No. 10/906,303, filedFeb. 14, 2005; International Application No. PCT/US04/43465, filed Dec.23, 2004; International Application No. PCT/US04/33359, filed Oct. 8,2004 and U.S. Ser. No. 11/131,551, filed May 17, 2005, the contents ofwhich are incorporated herein by reference).

This invention provides a composition for AntiMS, antianeurysm,antiasthmatic, antibradykinic, anticapillarihemorrhagic, anticephalagic,anticervicobrachialgic, antieclamptic, antiedemic, antiencaphalitic,antiepiglottitic, antiexudative, antiflu, antifracture, antigingivitic,antihematomic, antiherpetic, antihistaminic, antihydrathritic,antimeningitic, antioxidant, antiperiodontic, antiphlebitic,antipleuritic, antiraucedo, antirhinitic, antitonsilitic, antiulcer,antivaricose, antivertiginous, cancerostatic, corticosterogenic,diuretic, fungicide, hemolytic, hyaluronidase inhibitor, lymphagogue,natriuretic, pesticide, pituitary stimulant, thymolytic, vasoprotective,and venotonic treatment.

A composition comprising an effective amount of the compound of any oneof Y0, Y1, Y2, Y, Y7, Y8, Y9, Y10, or a salt, ester, metabolite orderivative thereof as a medicament for inhibiting tumor or cancer cellgrowth and for treating cancer, wherein the cancers comprise breastcancer, leukocyte cancer, liver cancer, ovarian cancer, bladder cancer,prostate cancer, skin cancer, bone cancer, brain cancer, leukemiacancer, lung cancer, colon cancer, CNS cancer, melanoma cancer, renalcancer or cervix cancer.

This composition can be administered orally or in a particularembodiment, it can be administered through intraperitoneal (I.P.),intravenous (I.V.) injection or intravenous drip. In an embodiment, themedicine can be administered with glucose solution or NaCl solution. Theadministration of the medicine can be as intravenous injection orintravenous drip. Example 1: Intravenous drip: 0.05-0.2 mg/kg medicinedissolved in 250 ml of 10% glucose solution or in 250 ml of 0.9% NaClsolution. Example 2: Intravenous injection: 0.05-0.2 mg/kg/day medicinedissolved in 10-20 ml of 10% glucose solution or of 0.9% NaCl solution.Course of treatment: 7-10 days.

Example 3

Intravenous drip: 0.1-0.2 mg/kg/day medicine dissolved in 250 ml of 10%glucose solution or in 250 ml of 0.9% NaCl solution. Course oftreatment: 7-10 days.

Example 4

Intravenous injection: 0.1-0.2 mg/kg/day medicine dissolved in 10-20 mlof 10% glucose solution or of 0.9% NaCl solution. Course of treatment:7-10 days.

Example 5

Intraperitoneal (I.P.): 2.5 mg/kg/day medicine dissolved in 10% glucosesolution or of 0.9% NaCl solution. Course of treatment: 7-10 days.

The composition can be administered orally wherein the dosage of mammalis 1-10 mg/Kg. The composition can be administered orally wherein thedosage is 10-30 mg/Kg. The composition can be administered orallywherein the dosage is 30-60 mg/Kg. The composition can be administeredorally wherein the dosage is 60-90 mg/Kg. The composition can beadministered intravenous injection or intravenous drip wherein thedosage of mammal is 0.01-0.1 mg/Kg. The composition can be administeredintravenous injection or intravenous drip wherein the dosage is 0.1-0.2mg/Kg. The composition can be administered intravenous injection orintravenous drip wherein the dosage is 0.2-0.4 mg/Kg. The compositioncan be administered intravenous injection or intravenous drip whereinthe dosage is 0.4-0.6 mg/Kg. The composition can be administeredintraperitoneal (I.P.) wherein the dosage of mammal is 1-3 mg/Kg. Thecomposition can be administered intraperitoneal (I.P.) wherein thedosage is 3-5 mg/Kg. The composition can be administered intraperitoneal(I.P.) wherein the dosage is 4-6 mg/Kg. The composition can beadministered intraperitoneal (I.P.) wherein the dosage is 6-10 mg/Kg.This invention provides a method of treating a mammal for treatingcancers; for inhibiting virus; for preventing cerebral aging; forimproving memory; improving cerebral functions, for curing enuresis,frequent micturition, urinary incontinence, dementia, Alzheimer'sdisease, autism, brain trauma, Parkinson's disease or other diseasescaused by cerebral dysfunctions; for treating arthritis, rheumatism,poor circulation, arteriosclerosis, Raynaud's syndrome, angina pectoris,cardiac disorder, coronary heart disease, headache, dizziness, kidneydisorder; cerebrovascular diseasea; inhibiting NF-Kappa B activation;for treating brain edema, sever acute respiratory syndrome, respiratoryviral diseases, chronic venous insufficiency, hypertension, chronicvenous disease, anti-oedematous, anti inflammatory, hemonhoids,peripheral edema formation, varicose vein disease, flu, post traumaticedema and postoperative swelling; for inhibiting ethanol absorption; forlowering blood sugar; for regulating the adrenocorticotropin andcorticosterone level comprising administering to said mammal atherapeutically effective amount of a pharmaceutical compositioncomprising a composition comprises the molecular formula or compound inthis invention.

This invention provides a method of treating a mammal for treatingcancers comprising administering to said mammal a therapeuticallyeffective amount of a pharmaceutical composition comprising acomposition comprises the molecular formula or compound in thisinvention. The cancers are included but not limited to:

Leukemia cancer, Lung cancer, Colon cancer, CNS cancer, Melanoma cancer,Ovarian cancer, Renal cancer, Prostate cancer, Breast cancer, bladdercancer, cervix cancer, liver cancer, bone cancer, brain cancer and Skincancer. The compounds comprise Xanifolia Y0, Y1, Y2, Y, Y7, Y8, Y9, Y10,or a salt, ester, metabolite or derivative thereof. This inventionprovides a method comprising the compounds interacting with cancer cellsand increases the static charge of the cells, which increase water flowinto the cells. The cancer cells are collapsed. The compounds compriseXanifolia Y0, Y1, Y2, Y, Y7, Y8, Y9, Y10, X or a salt, ester, metaboliteor derivative thereof. This invention also provides a method fortreating cancers comprising administering to said mammal atherapeutically effective amount of a pharmaceutical compositioncomprising a composition comprises the molecular formula or compound inthis invention. The cancers comprise Leukemia cancer, Lung cancer, Coloncancer, CNS cancer, Melanoma cancer, Ovarian cancer, Renal cancer,Prostate cancer, Breast cancer, bladder cancer, cervix cancer, livercancer, bone cancer, brain cancer and Skin cancer. The compoundscomprise Xanifolia Y0, Y1, Y2, Y, Y7, Y8, Y9, Y10, or a salt, ester,metabolite or derivative thereof.

This invention describes a method interacting or regulating the proteinon the surface of a cell or altering the functional properties ofintracellular membranes or regulating the fluid passage through the cellwall to kill the cancer cells. The method comprising administeringcontacting an effective amount of compound is selected from formula (1),(1A), (1B), (1C), (1D) preferable Xanifolia Y0, Y, Y1, Y2, Y7, Y8, Y9,Y10. In an embodiment, the compound is selected from Xanifolia Y0, Y,Y1, Y2, Y7, Y8, Y9, and Y10 interact with the protein in the membraneand open up the channel for water or solute particle. The cell takes inwater or solute particle and bursts. In an embodiment the components ofthe compound select from Xanifolia Y0, Y, Y1, Y2, Y7, Y8, Y9, and Y10combine with the protein in the membrane and open up the channel forwater or solute particle. The cell takes in water or solute particle andbursts.

One or more aquaporin AQPs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 in the cancercell membrane is overexpressed. So as providing more chance react withXanifolia compound. Water or ion particle pass through the cell membranethe cancer cells. The compound is selected from Xanifolia Y0, Y, Y1, Y2,Y7, Y8, Y9, and Y10 dilute the solution outside the cancer to make morewater pass in the cell. The overexpress of Aquaporin of AQPs 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 provide more channel for water or ion particle whichcauses cancer cell die. This invention provides a method of inhibitingcancer growth by destroys the cancer cell wherein aquaporin isoverexpressed; wherein the cancers comprise breast cancer, leukocytecancer, liver cancer, ovarian cancer, bladder cancer, prostate cancer,skin cancer, bone cancer, brain cancer, leukemia cancer, lung cancer,colon cancer, CNS cancer, melanoma cancer, renal cancer or cervixcancer. In an embodiment, the cancer is ovarian cancer. This inventionprovides a method of treating a mammal for treating cancers comprisingadministering to said mammal a therapeutically effective amount of apharmaceutical composition comprising a composition comprises themolecular formula or compound in this invention. The cancers areincluded but not limited to Leukemia cancer, Lung cancer, Colon cancer,CNS cancer, Melanoma cancer, Ovarian cancer, Renal cancer, Prostatecancer, Breast cancer, bladder cancer, cervix cancer, liver cancer, bonecancer, brain cancer and Skin cancer. The compounds comprise XanifoliaY0, Y1, Y2, Y, Y7, Y8, Y9, Y10, or a salt, ester, metabolite orderivative thereof.

To investigate the anti-tumor activity of Xanthoceras sorbifolia, weemployed cancer cell lines derived from different human organs andtested the effect on growth activity. In these preliminary studies, wefound that the plant extract inhibits the growth of certain cell lines.We studied 10-15 cell-lines (derived from different human organs) with aMTT cell-growth assay, and found that OVCAR3 cells (from ovary) to bethe most sensitive (with IC50=14.5 ug/ml). Int'l App'l No.PCT/US04/33359 and U.S. Ser. No. 10/906,303. The active compound wasthen purified and named Xanifolia-Y. Its chemical structure wasdetermined by 2D NMR and MS analysis. Xanifolia-Y is a noveltriterpenoid saponin with a diangeloyl group attached at one end andcarbohydrates or sugar moieties at another end of the triterpenestructure. In an embodiment, the diangeloyl attached at C21, C22positions and carbohydrates or sugar moieties at C3 position of atriterpene structure. The diangeloyl group is important for itsactivity. The purified compound has been tested with 60 cancer celllines. Test results show inhibition towards most cell lines tested withGI50 values ranging from 0.1-1 uM.

OVCAR3 cells, cancer cells derived from ovary, are the most sensitive toXanifolia-Y among cell lines tested in our early studies. Wesubsequently tested 10 additional human ovarian cancer cell lines andfound all of them to be susceptible to inhibition by Xanifolia-Y withIC50 values ranging from 2-12 uM.

See experiment 10 In vivo studies employing human ovarian carcinomaxenografts in nude mice were performed. The human ovarian cancer cells(ES2) were inoculated into the peritoneal cavity of nude mice andsubsequently received Xanifolia-Y. (Experiment 7, 8, 9). The tumorbearing mice received the drug (by i.p. route) for 10 days starting fromeither day 1, day 4, or day 10 after inoculations. The results show thatthe median survival time for tumor bearing mice without drug-treatmentis approximately 20-24 days. However, there was no death for tumorbearing mice with drug-treatment starting on day 1 after tumorinoculation. The median survival time for tumor bearing mice withdrug-treatment starting on day 4 after tumor inoculation is no death in50 days; and tumor bearing mice with drug-treatment started on day 10after tumor inoculation is half of the mice survive in 50 days. Theseresults indicate that the compounds of this invention are capable ofincreasing the survival rate of mammal. The median survival time fortumor bearing mice with drug-treatment starting on day 4 after tumorinoculation is 58 days (extension of life span of 141%); and tumorbearing mice with drug-treatment started on day 10 after tumorinoculation is 31 days (extension of life span of 29%). These resultsindicate that Xanifolia-Y is capable of extending the life span ofmammal bearing tumors. It is useful in treating ovarian cancer inhumans. These results indicate that Xanifolia-Y is capable of extendingthe life span of mammal bearing tumors. It is useful in treating ovariancancer in humans.

Among gynecological malignancies, ovarian cancer has the highest rate ofmortality in women in the United States with an estimated 22,220 newcases in 2005 and over 16,000 deaths (NIH web info). The disease isoften missed in diagnosis in the early stage due to asymptomatic and thelack of reliable diagnostic marker. As a result, most of the ovariancancer patients being diagnosed are already at advanced stages. Thestandard treatment of ovarian cancer is a combination of a platinumanalogue with paclitaxel (McGuire et al., 1996; Ozols et al., 2003).Improved patient survival time was observed in patients withintraperitoneal administration of these agents (Armstrong et al., 2006).The peritoneal cavity is the principal site of disease in ovariancancer. The improved efficacy of these agents could be due to a moredirect interaction with cancer cells. However, the increase of mediansurvival from 49.7 to 65.6 months is still far from satisfactory. Asmentioned above, our in vivo animal experiments mimicking the humansituation showed that Xanifolia-Y is effective in prolonging mammal lifespan. Mice were inoculated with human ovarian carcinoma (ES2) in theperitoneal cavity. Starting from the mid-way (time to mortality) pointof tumor progression (considered as a late stage of disease in human),drug was then administered into the peritoneal cavity. It was found thatXanifolia-Y treatment is beneficial to tumor bearing mice by prolongingtheir life span. Depending on the stage of the disease progression, thesooner the start of the drug-treatment, the better the results are.Based on our results, it can prolong the life-span of tumor bearing miceafter Xanifolia-Y-treatment is due to blockage of the migration ormetastasis of inoculated cancer cells into the mesothelium lining in theperitoneal cavity. In vitro studies show that Xanifolia-Y inhibits celladhesion to culture flasks (See Experiment 38). It is known thatadhesive molecules play an important role in the migration andmetastasis of ovarian cancer (Skubitz, 2002, Schaller, 1996; Zetter,1993). A major route for the spread of ovarian cancer is by theattachment of tumor cells to the mesothelium lining in the peritonealcavity (Gardner et al., 1995). Xanifolia-Y blocks the function of theseadhesive molecules on cells. In an embodiment, Xanifolia-Y blocks thefunction of these adhesive molecules on carcinoma cells. In anembodiment, Xanifolia-Y blocks the function of these adhesive moleculeson ovarian carcinoma cells. In an embodiment, Xanifolia-Y blocks thefunction of these adhesive molecules on the mesothelial cells. In anembodiment, Xanifolia-Y binds to the adhesive proteins (by masking) onthe membrane and inhibits the interaction of adhesion proteins withtheir receptors. In an embodiment, Xanifolia-Y action on membraneaffects adhesion proteins' function in membrane. The lost of adhesionactivity of cancer cells is result from direct or indirect action ofXanifolia-Y on membrane proteins. Most of the adhesion proteins areglycoproteins. The carbohydrate moiety in adhesion proteins interactwith carbohydrates from other molecules, such as saponin or Xanifolia-Y.Xanifolia-Y has a trisaccharide at the C3 position and it was found thata loss of carbohydrates reduces its activity (FIGS. 3D and 4C). Our EMstudies show that Xanifolia-Y affects membrane structure and makesholes. Damage to the membrane structure could alter adhesion protein'sconformation and interfere with their binding with other molecules oreven cause them to lose their anchorage on membrane. Our studies ofXanifolia-Y indicate it can be used in cancer therapy, especially as abenefit to patients with late stage ovarian cancer. They indicate thatour determined saponins and formulas are useful in cancer therapy bydemonstrating its inhibition of tumor growth in mammal systems.

We labeled Xanifolia-Y as ligands and used it to confirm its bindings,such as its location on cells, binding to adhesion proteins or othertarget protein with RIA, investigate its associated proteins with co-IPand verify them with competition assay. We confirm Xanifolia-Y as ananticancer agent. Specifically, we determine tumor nodule growth inperitoneal cavity during Xanifolia-Y treatment.

Xanifolia-Y blocks cancer migration and metastasis. In an embodiment, itblocks ovarian cancer migration and metastasis. Xanifolia-Y has effecton membrane and adhesion proteins.

To study the effect of Xanifolia-Y on membrane structure, the morphologyof cell membrane treated with Xanifolia-Y was examined with EM. In thisexperiment, K562 cells were treated with 5 uM of Xanifolia-Y for 60 min.Solvent DMSO and AKOH-Y (a derivative of Xanifolia-Y without theangeloyl group and it has no activity) served as controls. Cells werenegative stained with 1% UAc and subsequently examined with EM. FIG. 27show that patches of pits were found in the membrane of Xanifolia-Ytreated cells (FIG. 27B) but not in cells treated with the DMSO (FIG.27A) or AKOH-Y (FIG. 27C) controls. These pits have the size from 80 Ato 500 A (in diameter). The pits represent holes formed in the membrane.The pits are arranged in a characteristic pattern with smaller pits (80A in diameter) located in the periphery and the bigger ones (500 A indiameter) in the center. The bigger holes are resulted from fusing ofthe smaller holes (FIG. 27D). Membrane image of cells treated with A:DMSO solvent control, 60 min (magnification: ×60,000); B: Xanifolia-Y 5uM, 60 min. (×60000); C: AKOH-Y, 20 uM, 60 min. (×60000); D: Xanifolia-Y5 uM, 60 min. (×20000). This experiments results show that theXanifolia-Y alters the membrane of cell. In an embodiment, it damagesthe membrane of cancer cell. See FIG. 27.

Xanifolia-Y can be used for inhibiting cancers cell growth or treatingcancers wherein the cancers comprise breast cancer, leukocyte cancer,liver cancer, ovarian cancer, bladder cancer, prostate cancer, skincancer, bone cancer, brain cancer, leukemia cancer, lung cancer, coloncancer, CNS cancer, melanoma cancer, renal cancer or cervix cancer,wherein the cancer is preferably ovarian cancer. Among the differentcell lines tested in our studies, carcinoma cells derived from ovaryproved to be the most sensitive, a finding which is substantiated withmore human ovarian cancer cell lines. The results of animal studies withhuman tumor xenograft in mice show that it can extend the life span ofmice bearing tumors. The compounds of this application can extend thelife span of mammal bearing human cancer. Cancer drugs that target onmembrane or membrane constituents are not explored. Xanifolia-Y is a newdrug. It has effects on cell membrane, a target that differs fromcurrent anticancer drugs.

This invention provides a method of altering the characteristic ofcancer cell membrane to block the migration, metastasis of cancer cellsor inhibit the growth of cancers or anti-angiogenesis.

This invention provides a method of inhibiting the growth, migration,metastasis of cancer by altering the characteristic of membrane ofcancer cell, wherein the characteristic comprise adhesion protein;wherein the cancers comprise breast cancer, leukocyte cancer, livercancer, ovarian cancer, bladder cancer, prostate cancer, skin cancer,bone cancer, brain cancer, leukemia cancer, lung cancer, colon cancer,CNS cancer, melanoma cancer, renal cancer or cervix cancer, wherein themethod is administering contacting Xanifolia Y0, Y1, Y2, Y, Y7, Y8, Y9,Y10, or a salt, ester, metabolite thereof.

This invention provides a method of inhibiting the growth, migration,metastasis of cancer by altering the adhesion characteristic of membraneof cancer cell, wherein the cancers comprise breast cancer, leukocytecancer, liver cancer, ovarian cancer, bladder cancer, prostate cancer,skin cancer, bone cancer, brain cancer, leukemia cancer, lung cancer,colon cancer, CNS cancer, melanoma cancer, renal cancer or cervixcancer, wherein the method is administering contacting Xanifolia Y0, Y1,Y2, Y, Y7, Y8, Y9, Y10, or a salt, ester, metabolite thereof. In anembodiment the method is administering contacting the compound selectedfrom formula in this application.

This invention provides a composition for inhibiting the growth,migration, metastasis of cancer by altering the adhesion characteristicof membrane of cancer cell, wherein the cancers comprise breast cancer,leukocyte cancer, liver cancer, ovarian cancer, bladder cancer, prostatecancer, skin cancer, bone cancer, brain cancer, leukemia cancer, lungcancer, colon cancer, CNS cancer, melanoma cancer, renal cancer orcervix cancer. This application shows Xanifolia-Y is an alternate orsupplemental agent to DNA-inhibition or microtubule-targeting drugs. Itcould be beneficial if it is used singly or in combination with otherdrugs of different mechanisms (block M-phase progression or DNAsynthesis). Our inventions show combined effect of Xanifolia-Y andpaclitaxel on inhibition of ES2 cells' growth (Detail in Experiment 15)

Identify the binding target of Xanifolia-Y of adhesion proteins andsignaling proteins in ovarian cancer cells. In our animal studies, itwas shown that Xanifolia-Y extended the life span of tumor bearing mice.(See Experiments 7, 8, 9). The animals died sooner if the treatment ofXanifolia-Y was delayed (comparing results of treatments started from 1,4 or 10 days after tumor inoculation). The results show that Xanifolia-Yinhibits migration or metastasis of the inoculated cancer cells. Ovariancarcinoma cells express high levels of adhesion molecules. Adhesionproteins are present in both cancer cells and mesothelial cells. Whilethe lost of adhesion is blocking of the protein accessibility due todirect binding to Xanifolia-Y. In an embodiment, the interaction ofXanifolia-Y with membrane alter indirectly the adhesion protein'sbinding site(s).

We have shown that Xanifolia-Y are cytotoxic to tumor cells, In anembodiment, it kills ovarian cancer cells. Our inventions show thatXanifolia-Y inhibits cancer cell growth and prolongs life-span of tumorbearing mice. Our studies also indicate that the sooner thedrug-treatment, the longer the life-span of the tumor bearing animals isextended. Xanifolia-Y also has an effect in blocking or inhibitingmigration or metastasis. The delay of Xanifolia-Y-treatment allows morechances for cancer cells to metastasize to the mesothelium lining in theperitoneal cavity which resulted in more tumor growth and shorter lifespan. Adhesive molecules play an important role in cell migration andmetastasis. It was shown in our studies that Xanifolia-Y inhibits cellattachment to culture flasks. Xanifolia-Y interferes with the functionof the adhesive molecules. In embodiment Xanifolia-Y blocks the functionof the adhesive molecules. In an embodiment, Xanifolia-Y binds directlyto adhesive proteins. It is masking the adhesive proteins. In anembodiment, Xanifolia-Y indirectly alters membrane structure that causechanges in protein conformation, or locations and result in loss ofadhesion process.

This invention provides methods and compositions for modulating geneexpression to cure diseases or reduce the syndrome of diseases, whereinthe modulating comprises positive and negative regulating. In anembodiment, the method comprises inhibiting gene expression. In anembodiment the method comprises stimulating gene expression. Thisinvention provides methods and compositions for inhibiting themigration, metastasis or growth of cancers or anti-angiogenesis, whereinthe methods comprise affecting the gene expression, wherein the methodcomprises affecting adhesion proteins or their receptors, reducingadhesion proteins, or inhibiting the expression or secretion of adhesionproteins, wherein the adhesion proteins comprise fibronectin, integrinsfamily, myosin, vitronectin, collagen, laminin, glycosylation cellsurface proteins, polyglycans, cadherin, heparin, tenascin, CD 54, CAM,elastin and FAK.

This invention provides methods and compositions for inhibiting themigration, metastasis or growth of cancers or anti-angiogenesis, whereinthe methods comprise affecting the gene expression, wherein the methodcomprises stimulating gene expression. This invention provides a methodfor altering the characteristics of cancer cell membranes resulting inblocking the migration, metastasis of cancer cells or inhibiting thegrowth of cancers or anti-angiogenesis, wherein the method comprisesreducing adhesion proteins or their receptors, wherein the adhesionproteins comprise fibronectin, integrins family, myosin, vitronectin,collagen, laminin, glycosylation cell surface proteins, polyglycans,cadherin, heparin, tenascin, CD 54, CAM, elastin and FAK.

This invention provides methods, processes, compounds and compositionsof reducing expression or secretion of adhesion proteins of cells,wherein the adhesion proteins comprise fibronectin, integrins family,myosin, vitronectin, collagen, laminin, glycosylation cell surfaceproteins, polyglycans, cadherin, heparin, tenascin, CD54, CAM, elastinand FAK. In an embodiment, methods comprise inhibiting gene expression.In an embodiment, this invention provides a method of reducing thesecretion of fibronectin. In an embodiment the method can block themigration, metastasis of cancer cells or inhibit the growth of cancersor anti-angiogenesis, wherein the cancers comprise breast, leukocyte,liver, ovarian, bladder, prostate, skin, bone, brain, leukemia, lung,colon, CNS, melanoma, renal, cervix, esophagus, testis, spleen, kidney,lymph, pancreas, stomach and thyroid cancer. In an embodiment, themethod comprises contacting the cell with compound selected from Mb1,Mb2, Mb2.1, Mb3, Mb4, Mb5, Mb6, Mb7, Mb8, Mb9, Mb10, Mb11, Mb12 andMb13, ACH-Z4, ACH-Y10, ACH-Y2, ACH-Y8, ACH-Y7, ACH-Y0, ACH-X, ACH-E,Ba1, Ba2, Ba3, Ba4, Ba5, Ba6, Ba7, Ba8, Ba9, Ba10, Ba11, Ba12, Ba13,Ba14, Ba15, Ba16, Ba17, Xanifolia Y0, Y1, Y2, Y, Y5, Y7, Y8, Y9, Y10,Xanifolia (x), Escin or Aescin or a salt, ester, metabolite thereof andCompound A to X and A1 to X1 in the application.

This invention provides a method of altering the characteristics ofcancer cell membranes, wherein the method comprises altering thesecretion of adhesion proteins, wherein the adhesion proteins comprisefibronectin, integrins family, myosin, vitronectin, collagen, laminin,glycosylation cell surface proteins, polyglycans, cadherin, heparin,tenascin, CD 54, CAM, elastin and FAK. In an embodiment, the methods,processes, compounds and compositions comprise blocking, suppressing orinhibiting the expression or secretion of adhesion protein, wherein theadhesion proteins comprise fibronectin, integrins family, myosin,vitronectin, collagen, laminin, glycosylation cell surface proteins,polyglycans, cadherin, heparin, tenascin, CD 54, CAM, elastin and FAK.In an embodiment, the methods, processes, compounds and compositions isinteracting with adhesion proteins, wherein the adhesion proteinscomprise fibronectin, integrins family, myosin, vitronectin, collagen,laminin, glycosylation cell surface proteins, polyglycans, cadherin,heparin, tenascin, CD 54, CAM, elastin and FAK. In an embodiment themethods, processes, compounds or compositions can block the migration,metastasis of cancer cells or inhibit the growth of cancers oranti-angiogenesis, wherein the cancers comprise cancers of breast,leukocyte, liver, ovarian, bladder, prostate, skin, bone, brain,leukemia, lung, colon, CNS, melanoma, renal, cervix, esophagus, testis,spleen, kidney, lymph, pancreas, stomach and thyroid. In an embodiment,the method comprises contacting the cell with compound selected fromMb1, Mb2, Mb3, Mb4, Mb5, Mb6, Mb7, Mb8, Mb9, Mb10, Mb11, Mb12 and Mb13,ACH-Z4, ACH-Y10, ACH-Y2, ACH-Y8, ACH-Y7, ACH-Y0, ACH-X, ACH-E, Ba1, Ba2,Ba3, Ba4, Ba5, Ba6, Ba7, Ba8, Ba9, Ba10, Ba11, Ba12, Ba13, Ba14, Ba15,Ba16, Ba17, Xanifolia Y0, Y1, Y2, Y, Y7, Y8, Y9, Y10, Xanifolia (x),Escin or Aescin or a salt, ester, metabolite thereof and Compound A to Xand A1 to X1 in the application.

The adhesion proteins help cancer cell adhesion, invasion or metastasis,wherein the cancers comprise ovarian cancer. Reducing the adhesionproteins will reduce the metastasis of cancers. Fibronectin is one ofthe key factors in the biology of epithelial ovarian cancers. Reductionof fibronectin will inhibit the metastasis of cancer cells.

This invention provides a method and composition for inhibiting theexpression or secretion of adhesion proteins comprising fibronectin inorder to cure diseases, wherein the diseases comprise inhibiting cancergrowth, wherein the cancers comprise breast, leukocyte, liver, ovarian,bladder, prostate, skin, bone, brain, leukemia, lung, colon, CNS,melanoma, renal, cervix, esophagus, testis, spleen, kidney, lymph,pancreas, stomach and thyroid cancer. In an embodiment, the methodcomprises contacting the cell with compound selected from Mb1, Mb2, Mb3,Mb4, Mb5, Mb6, Mb7, Mb8, Mb9, Mb10, Mb11, Mb12 and Mb13, ACH-Z4,ACH-Y10, ACH-Y2, ACH-Y8, ACH-Y7, ACH-Y0, ACH-X, ACH-E, ACH-Mb5,ACH-Mb13, Ba1, Ba2, Ba3, Ba4, Ba5, Ba6, Ba7, Ba8, Ba9, Ba10, Ba11, Ba12,Ba13, Ba14, Ba15, Ba16, Ba17, Xanifolia Y0, Y1, Y2, Y, Y7, Y8, Y9, Y10,Xanifolia (x), Escin or Aescin or a salt, ester, metabolite thereof andCompound A to X and A1 to X1 in the application.

This invention provides a use of compound or a composition formanufacture of medicament for inhibiting the growth, migration,metastasis of cancer or altering the characteristics of membranes ofcancer cell, wherein the characteristics comprise adhesion of proteins;wherein comprising the secretion of proteins or the adhesion of cells;wherein the characteristics comprise adhesion ability; wherein theadhesion proteins comprise fibronectin, integrins family, myosin,vitronectin, collagen, laminin, glycosylation cell surface proteins,polyglycans, cadherin, heparin, tenascin, CD 54, CAM, elastin and FAK;wherein the cancers comprise cancers of breast, leukocyte, liver,ovarian, bladder, prostate, skin, bone, brain, leukemia, lung, colon,CNS, melanoma, renal, cervix, esophagus, testis, spleen, kidney, lymph,pancreas, stomach and thyroid; wherein the method comprisesadministering to the subject or contacting the cells with the extract,compositions, saponins or compounds from Xanthoceras Sorbifolia,Harpullia, Aesculus hippocastanum, Maesa balansae and Barringtoniaacutangula for inhibiting cancer metastasis, wherein the cancerscomprise cancers of breast, leukocyte, liver, ovarian, bladder,prostate, skin, bone, brain, leukemia, lung, colon, CNS, melanoma,renal, cervix, esophagus, testis, spleen, kidney, lymph, pancreas,stomach and thyroid cancers; wherein extracts, compositions, saponins orcompounds are prepared from the husks, branches, stems, leaves, kernels,roots, barks, fruit, seeds or seed shells of the herb or plant.

The present invention provides vaccines for cancer immunotherapy. Thevaccines comprise extract, compositions, compounds and saponins fromXanthoceras Sorbifolia, Harpullia, Aesculus hippocastanum, Maesabalansae and/or Barringtonia acutangula. In embodiment, the compoundscan be obtained from synthesis, semi-synthesis or modification. Themethod comprises administering to the subject an effective amount ofvaccine for enhancing the immune response. The vaccines comprisesaponins isolated from Xanthoceras Sorbifolia, Harpullia, Aesculushippocastanum, Maesa balansae and/or Barringtonia acutangula.

The present invention provides adjuvant compositions for cancer curing.The adjuvant compositions comprise extract, compositions, compounds andsaponins from Xanthoceras Sorbifolia, Harpullia, Aesculus hippocastanum,Maesa balansae and/or Barringtonia acutangula. The method comprisesadministering to the subject an effective amount of the above adjuvantcompositions for enhancing the immune response. The use of vaccinecompositions comprises inhibiting cancer metastasis, wherein the cancerscomprise breast, leukocyte, liver, ovarian, bladder, prostate, skin,bone, brain, leukemia, lung, colon, CNS, melanoma, renal, cervix,esophagus, testis, spleen, kidney, lymph, pancreas, stomach and thyroidcancers.

The present invention provides a use of compound for manufacture ofmedicament or methods for making a vaccine, wherein the vaccinecomprises compounds or saponins from Xanthoceras Sorbifolia, Harpullia,Aesculus hippocastanum, Maesa balansae and/or Barringtonia acutangula,wherein the vaccine can be used for inhibiting cancer growth, whereinthe vaccine is having immune adjuvant activity, wherein the saponinscomprise Xanifolia Y0, Y1, Y2, Y, Y5, Y7, Y8, Y9, Y10, Xanifolia (x),Escin or Aescin or a salt, ester, metabolite thereof. In an embodimentthe saponin may be selected from formulas (1A), (1B), (1C), (1D), (1E),(1F), (1G), (1H), (1J), (1K), (1L). In an embodiment the compound(s) areselected from Compound Z1 to Z13 in the application. In an embodimentthe saponin(s) comprise Mb1, Mb2, Mb3, Mb4, Mb5, Mb6, Mb7, Mb8, Mb9,Mb10, Mb11, Mb12 and Mb13. In an embodiment, the compound comprises atriterpene backbone, two angeloyl groups and sugar moiety. In anembodiment the compound(s) are selected from Compound A to X and A1 toX1 in the application. In an embodiment the compound(s) are selectedfrom ACH-Z4, ACH-Y10, ACH-Y2, ACH-Y8, ACH-Y7, ACH-Y0, ACH-X, ACH-E,ACH-Mb5, and ACH-Mb12. In an embodiment the saponins comprise Ba1, Ba2,Ba3, Ba4, Ba5, Ba6, Ba7, Ba8, Ba9, Ba10, Ba11, Ba12, Ba13, Ba14, Ba15,Ba16 and Ba17. In an embodiment the compound(s) are selected fromXanifolia Y0, Y1, Y2, Y, Y5, Y7, Y8, Y9, Y10, Xanifolia (x), Escin orAescin or a salt, ester, metabolite thereof. This invention provides amethod of treating protozoal infections. In an embodiment, thisinvention provides a method for treating parasites by using the abovecompounds, wherein the method comprises inhibiting leishmaniases,amoebiasis, trypanosomiasis, toxoplasmosis or malaria; wherein themethod comprises contacting cells with an effective amount of anisolated, purified or synthesized compound, or its salt, or esterthereof, selected from the above compounds.

This invention provides a use of compound for manufacture of medicamentor a method for pharmaceutical composition useful for inducing an immuneresponse to an antigen in an individual comprising a saponin compositionfrom Xanthoceras Sorbifolia, Harpullia, Aesculus hippocastanum, Maesabalansae or Barringtonia acutangula or synthesis. The present inventionprovide methods for enhancing an immune response to an antigen in anindividual comprising administering an effective amount ofsaponins/compositions from Xanthoceras Sorbifolia, Harpullia, Aesculushippocastanum, Maesa balansae and/or Barringtonia acutangula, orsaponins comprising Xanifolia Y0, Y1, Y2, Y, Y5, Y7, Y8, Y9, Y10,Xanifolia (x), Escin or Aescin or a salt, ester, metabolite thereof, orthe saponin may be selected from formulas (1A), (1B), (1C), (1D), (1E),(1F), (1G), (1H), (1J), (1K), (1L). In an embodiment the compound(s) areselected from Compound Z1 to Z13 in the application. In an embodimentthe saponin comprise Mb1, Mb2, Mb3, Mb4, Mb5, Mb6, Mb7, Mb8, Mb9, Mb10,Mb11, Mb12 and Mb13. In an embodiment the compound(s) are selected fromCompound A to X and A1 to X1 in the application. In an embodiment thecomposition(s) are selected from ACH-Z4, ACH-Y10, ACH-Y2, ACH-Y8,ACH-Y7, ACH-Y0, ACH-X, ACH-E, ACH-Mb5, and ACH-Mb12. In an embodimentthe saponins comprise Ba1, Ba2, Ba3, Ba4, Ba5, Ba6, Ba7, Ba8, Ba9, Ba10,Ba11, Ba12, Ba13, Ba14, Ba15, Ba16, Ba17.

This invention provides methods or a use of compound for manufacture ofmedicament for modulating adhesion or angiogenesis of cancer cells,antiparasitics, enhancing an immune response, providing adjuvantactivities or providing vaccine activities, inhibiting cancer metastasisor growth, wherein the cancers comprise breast cancer, leukocyticcancer, liver cancer, ovarian cancer, bladder cancer, prostatic cancer,skin cancer, bone cancer, brain cancer, leukemia cancer, lung cancer,colon cancer, CNS cancer, melanoma cancer, renal cancer, cervicalcancer, esophageal cancer, testicular cancer, spleenic cancer, kidneycancer, lymphatic cancer, pancreatic cancer, stomach cancer and thyroidcancer; In an embodiment the method comprises modulating thephosphatidylinositol signaling system and regulating the gene expressionof RGS4, LEPR, ICFBP3, ANGPT2, GPNMB, NUPR1 or LOC100126784. Thecompounds can be purified from natural resource comprising XanthocerasSorbifolia, Harpullia, Aesculus hippocastanum, Maesa balansae orBarringtonia acutangula, or synthesized. The compounds comprise thefollowing:

(Our purification methods and biological assays including the MTT assayin International Application No. PCT/US05/31900, filed Sep. 7, 2005,U.S. Ser. No. 11/289,142, filed Nov. 28, 2005, and U.S. Ser. No.11/131,551, filed May 17, 2005, and PCT/US2008/002086, 1188-ALA-PCT,filed Feb. 15, 2008, the contents of which are incorporated herein byreference)

wherein R1 is O(C═O)C(CH3)═CH(CH3), R2 is (E) O(C═O)CH═CH—C6H5, R3 isOH, R4 is OH, also named Mb1; or wherein R1 is O(C═O)C(CH3)═CH(CH3), R2is (Z)—O(C═O)CH═CH—C6H5, R3 is OH, R4 is OH, also named Mb2; or whereinR1 is O(C═O)C(CH3)═CH(CH3), R2 is (Z)—O(C═O)C(CH3)═CH—C6H5, R3 is OH, R4is OH, also named Mb2.1; or wherein R1 is O(C═O)C6H5, R2 is(E)-O(C═O)CH═CH—C6H5, R3 is OH, R4 is OH, also named Mb3; or wherein R1is O(C═O)C6H5, R2 is (Z)—O(C═O)CH═CH—C6H5, R3 is OH, R4 is OH, alsonamed Mb4; or wherein R1, R2, are O(C═O)C(CH3)═CH(CH3), R3 is OH, R4 isOH, also named Mb5; or wherein R1 is O(C═O)C6H5, R2 isO(C═O)C(CH3)═CH(CH3), R3 and R4 are OH, also named Mb6; or

wherein R1 is O(C═O)C6H5, R2 is O(C═O)CH═CH—C6H5, R3, R4, R5 are OH, R6,R7, R8, R9, R10, R11, R12 are CH3, R13 is COOH also named Mb7; orwherein R1 is O(C═O)C6H5, R2 is O(C═O)CH═CH—C6H5, R3, R4, R5 are OH, R6is CH2OH, R7, R8, R9, R10, R11, R12 are CH3, R13 is COOH also named Mb8;or wherein R1 is O(C═O)C(CH3)═CH(CH3), R2 is O(C═O)CH═CH—C6H5, R3, R4are OH, R6, R7, R8, R9, R10, R11, R12 are CH3, R13 is COOH also namedMb9; or wherein R1 is O(C═O)C(CH3)═CH(CH3), R2 is O(C═O)CH═CH—C6H5, R3,R4 are OH, R6 is CH2OH, R7, R8, R9, R10, R11, R12 are CH3, R13 is COOH,also named Mb10; or wherein R1, R2, are O(C═O)C(CH3)═CH(CH3), R3 is OH,R4 is CH2OH, R5 is H, R6, R7, R8, R9, R10, R11, R12 are CH3, R13 isCOOH, also named Mb11; or wherein R1 is O(C═O)C(CH3)═CH(CH3), R2 isO(C═O)CH═CH—C6H5, R3, R4 are OH, R6, R7, R8, R9, R10, R11, R12 are CH3,R13 is COOCH3, also named Mb12;or wherein R1 is O(C═O)C(CH3)═CH(CH3), R2 is (Z)—O(C═O)C(CH3)═CH—C6H5,R3 is OH, R4 is OH, R5 is H, and R6, R7, R8, R9, R10, R11, R12 are CH3,R13 is COOH, also named Mb13; or

3-O-[β-D-galactopyranosyl(1→2)]-β-D-xylopyranosyl(1→3)-β-D-glucuronopyranosyl-21-O-angeloyl, 22-O-angeloyl-3β, 15α, 21β,22α, 28-pentahydroxyolean-12-ene, named Ba 1

wherein R1 is O(C═O)C(CH3)═CH(CH3), R2 is O(C═O)C(CH3)═CH(CH3), R3 isCOOCH3, formula is 3-O-[β-D-galactopyranosyl(1→2)]-β-D-xylopyranosyl(1→3)-β-D-methylglucuronopyranosyl-21-O-angeloyl, 22-O-angeloyl-3β, 15α,21β, 22α, 28-pentahydroxyolean-12-ene, named Ba 2; or wherein R1 isO(C═O)C6H5, R2 is O(C═O)C(CH3)═CH(CH3), R3 is COOH, formula is:3-O-[β-D-galactopyranosyl(1→2)]-β-D-xylopyranosyl(1→3)-β-D-glucuronopyranosyl-21-O-benzoyl, 22-O-angeloyl-3β, 15α, 21β,22α, 28-pentahydroxyolean-12-ene, named Ba 3; or wherein R1 isO(C═O)C6H5, R2 is O(C═O)C(CH3)═CH(CH3), R3 is COOCH3, formula is:3-O-[β-D-galactopyranosyl(1→2)]-β-D-xylopyranosyl(1→3)-β-D-methylglucuronopyranosyl-21-O-benzoyl, 22-O-angeloyl-3β, 15α,21β, 22α, 28-pentahydroxyolean-12-ene, named Ba 4; or wherein R1 isO(C═O)C6H5, R2 is O(C═O)C6H5, R3 is COOH, formula is:3-O-[β-D-galactopyranosyl(1→2)]-β-D-xylopyranosyl(1→3)-β-D-glucuronopyranosyl-21-O-benzoyl, 22-O-benzoyl-3β, 15α, 21β,22α, 28-pentahydroxyolean-12-ene, named Ba 5; or wherein R1 isO(C═O)C6H5, R2 is O(C═O)C6H5, R3 is COOCH3, formula is:3-O-[β-D-galactopyranosyl(1→2)]-β-D-xylopyranosyl(1→3)-β-D-methylglucuronopyranosyl-21-O-benzoyl, 22-O-benzoyl-3β, 15α,21β, 22α, 28-pentahydroxyolean-12-ene, named Ba 6; or wherein R1 isO(C═O)C6H5, R2 is O(C═O)CH3CH3, R3 is COOH, formula is:3-O-[β-D-galactopyranosyl(1→2)]-β-D-xylopyranosyl(1→3)-β-D-glucuronopyranosyl-21-O-benzoyl, 22-O-isobutyryl-3β, 15α, 21β,22α, 28-pentahydroxyolean-12-ene, named Ba 7; or wherein R1 isO(C═O)C6H5, R2 is OH, R3 is COOH, formula is:3-O-[β-D-galactopyranosyl(1→2)]-β-D-xylopyranosyl(1→3)-β-D-glucuronopyranosyl-21-O-benzoyl-3β, 15α, 21β, 22α,28-pentahydroxyolean-12-ene, named Ba 8

wherein R1 is OH, R2 is O-benzoyl, R3 is O-benzoyl,3-O-[β-D-galactopyranosyl(1→2)]-β-D-xylopyranosyl(1→3)-β-D-glucuronopyranosyl-21-O-[3,4-dibenzoyl-3-D-xylopyranosyl]-3β,15α, 21β, 22α, 28-pentahydroxyolean-12-ene, named Ba9; R1 is O-acetyl,R2 is O-benzoyl, R3 is O-benzoyl3-O-[β-D-galactopyranosyl((1→2)]-β-D-xylopyranosyl(1→3)-β-D-gluouronopyranosyl-21-O-[3,4-dibenzoyl-β-D-xylopyranosyl]-22-O-acetyl-3β,15α, 21β, 22α, 28-pentahydroxyolean-12-ene, named Ba10;3-O-[β-D-galactopyranosyl(1→2)]-β-D-xylopyranosyl(1→3)-β-D-glucuronopyranosyl-21-O-[3-angeloyl,4-benzoyl-β-D-xylopyranosyl]-3β, 15α, 21β, 22α,28-pentahydroxyolean-12-ene, named Ba11;3-O-[β-D-galactopyranosyl(1→2)]-β-D-xylopyranosyl(1→3)-β-D-glucuronopyranosyl-21-O-[3,4-diangeloyl-β-D-xylopyranosyl]-3β,15α, 21β, 22α, 28-pentahydroxyolean-12-ene, named Ba12;3-O-[β-D-galactopyranosyl(1→2)]-β-D-xylopyranosyl(1→3)-β-D-glucuronopyranosyl-21-O-[3-angeloyl,4-tigloyl-β-D-xylopyranosyl]-3β, 15α, 21β, 22α,28-pentahydroxyolean-12-ene, named Ba13;

wherein R1 is OH, R2 is O-benzoyl, R3 is O-benzoyl3-O-[β-D-galactopyranosyl(1→2)]-β-D-xylopyranosyl(1→3)-β-D-gluouronopyranosyl-21-O-[3,4-dibenzoyl-α-L-arabinopyranosyl]-3β,15α, 21β, 22α, 28-pentahydroxyolean-12-ene, named Ba14; wherein R1 isO-acetyl, R2 is O-benzoyl, R3 is O-benzoyl3-O-[β-D-galactopyranosyl((1→2)]-β-D-xylopyranosyl(1→3)-β-D-gluouronopyranosyl-21-O-[3,4-dibenzoyl-α-L-arabinopyranosyl]-22-O-acetyl-3β,15α, 21β, 22α, 28-pentahydroxyolean-12-ene, named Ba15;

3-O-[β-D-galactopyranosyl(1→2)]-β-D-xylopyranosyl(1→3)-β-D-glucuronopyranosyl-21-O-[3-angeloyl-4-(3-benzoyl-2-methylbutyryl)-α-L-arabinopyranosyl]-22-O-acetyl-3β,15α, 21β, 22α, 28-pentahydroxyolean-12-ene, named Ba16;

3-O-[β-D-galactopyranosyl(1→2)]-β-D-xylopyranosyl(1→3)-β-D-glucuronopyranosyl-21-O-angeloyl, 22-O-angeloyl-3β, 15α, 16α,21β, 22α, 28-hexahydroxyolean-12-ene, also named Ba 17.Anti-Cancer Activities:

The anti-cancer activity of Mb's compounds with ES2 cell: the IC50 ofMb1 is 8 ug/ml, Mb2 is /ml, Mb3 is 8 ug/ml, Mb4 is 15 ug/ml, Mb5 is 6.5ug/ml, and Mb6 is 10 ug/ml, Mb7 is 12 ug/ml, Mb8 is 20 ug/ml, Mb9 is 18ug/ml, Mb12 is 10 ug/ml.

The anti-cancer activity of ACH's compounds with ES2 cell: the IC50 ofACH-Z4 is 40 ug/ml, ACH-Y3 is 20 ug/ml, ACH-Y10 is 20 ug/ml, ACH-Y2 is35 ug/ml, ACH-Y8 is 35 ug/ml, ACH-Y7 is 65 ug/ml, ACH-Y0 is 20 ug/ml,ACH-X is 40 ug/ml, ACH-E is 60 ug/ml

The anti-cancer activity of Ba compounds with ES2 cell: Ba1 is 5 ug/ml,Ba2 is 5 ug/ml, Ba3 is 8 ug/ml, Ba 5 is 16 ug/ml, Ba7 is 11 ug/ml, Ba8is 20 ug/ml, Ba9 is 12 ug/ml, Ba17 is 5 ug/ml.

The anti-cancer activity of Xanifolia Y with cells: the IC50 of Y on(bladder) TB9 cells is 5 ug/ml; IC50 of Y on (lung) H460 cells is 7.5ug/ml; IC50 of Y on HeLa cells is 20 ug/ml; IC50 of Y on skin cells is12 ug/ml; IC50 of Y on ES2 (ovarian) cells is 5 ug/ml; IC50 of pure Y on(Mouth) KB cells is 6 ug/ml;

IC50 of Z12 on ES2 (ovarian) cells is 16 ug/ml; Z4 is 20 ug/ml

IC50 of Mb5: (bladder) TB9 is 6.5 ug/ml, (Prostate) DU145 is 7.6 ug/ml,(Lung) H460 is 12 ug/ml (Liver) HepG2 is 6.5 ug/ml, (brain) T98G is 12ug/ml, (Skin) SK-MELS is 25 ug/ml, (Ovary) ES2 is 6.5 ug/ml, (Breast)MCF7 is 11 ug/ml.

IC 50 of ACH-Mb5: (bladder) TB9 is 5.7 ug/ml, (Prostate) DU145 is 6.4ug/ml, (Lung) H460 is 6.5 ug/ml (Liver) HepG2 is 4 ug/ml, (brain) T98Gis 6 ug/ml, (Skin) SK-MELS is 22 ug/ml, (Ovary) ES2 is 8 ug/ml, (Breast)MCF7 is 13 ug/ml.

This invention provides a method or composition for reducing theexpression and secretion of adhesion proteins to cure diseases, whereinthe diseases comprise cancer growth, leg swelling, symptoms of chronicvenous insufficiency, peripheral edema, lipemic, chronic venous disease,varicose vein disease, varicose syndrome, venous stasis, expectorant,peripheral vascular disorders, cerebro-organic convulsion, cerebralcirculation disorder, cerebral edema, psychoses, dysmenorrheal,hemorrhoids, episiotomies, peripheral edema formation or postoperativeswelling; leg pain; for pruritis, lower leg volume, symptoms of pain;thrombosis, thromophlebitis or for preventing gastric ulcers, spasms,comprising administering to a subject, in need thereof, an effectiveamount of the composition of this invention. In an embodiment, themethod comprises interacting with adhesion proteins, wherein theadhesion proteins comprise fibronectin, integrins family, myosin,vitronectin, collagen, laminin, glycosylation cell surface proteins,polyglycans, cadherin, heparin, tenascin, CD 54, CAM, elastin and FAK.In an embodiment, this invention provides a method of reducing thesecretion of fibronectin.

In an embodiment, the method comprises reducing the adhesion ability ofadhesion proteins; wherein the adhesion proteins comprise fibronectin,integrins family, myosin, vitronectin, collagen, laminin, glycosylationcell surface proteins, polyglycans, cadherin, heparin, tenascin, CD 54,CAM, elastin and FAK.

In an embodiment, the method comprises modulating the expression orsecretion of adhesion proteins, wherein the adhesion proteins comprisefibronectin, integrins family, myosin, vitronectin, collagen, laminin,glycosylation cell surface proteins, polyglycans, cadherin, heparin,tenascin, CD 54, CAM, elastin and FAK. In an embodiment, the methodcomprises blocking the secretion of adhesion proteins, wherein theadhesion proteins comprise fibronectin. In an embodiment the methodcomprises administering to a subject or contacting the cell with aneffective amount of the compound selected from formulas in thisapplication.

In an embodiment, this invention provides a method and composition formodulating adhesion or angiogenesis of cancer cells, antiparasitics ormanufacturing an adjuvant composition. In an embodiment, this inventionprovides a method of treating protozoal infections includingleishmaniases, amoebiasis, trypanosomiasis, toxoplasmosis and malariainfections.

This invention provides a use of compound for manufacture of medicament,a method and a composition for altering the characteristics of adhesionproteins to cure diseases, wherein the characteristics comprisingadhesion ability, wherein the method comprises reducing the secretion offibronectin, wherein the diseases comprise cancer growth, leg swelling,symptoms of chronic venous insufficiency, peripheral edema, lipemic,chronic venous disease, varicose vein disease, varicose syndrome, venousstasis, expectorant, peripheral vascular disorders, cerebro-organicconvulsion, cerebral circulation disorder, cerebral edema, psychoses,dysmenorrheal, hemorrhoids, episiotomies, peripheral edema formation orpostoperative swelling; symptoms of leg pain; pruritis, lower legvolume, symptoms of pain; thrombosis, thromophlebitis; for preventinggastric ulcers antispasmotic, comprising administering to a subject, inneed thereof, an effective amount of the composition of this invention;wherein the adhesion proteins comprise fibronectin, integrins family,myosin, vitronectin, collagen, laminin, glycosylation cell surfaceproteins, polyglycans, cadherin, heparin, tenascin, CD 54, CAM, elastinand FAK. In an embodiment the method is administering contacting aneffective amount in a subject with the compound selected from formulasin this application.

This invention provides a process and method for administration of thecomposition, wherein administration is by intravenous injection,intravenous drip, intraperitoneal injection or oral administration;wherein administration is by intravenous drip: 0.05-0.2 mg/kg compounddissolved in 250 ml of 10% glucose solution or in 250 ml of 0.9% NaClsolution, or by intravenous injection: 0.05-0.2 mg/kg/day compounddissolved in 10-20 ml of 10% glucose solution or of 0.9% NaCl solution,or by intravenous drip: 0.1-0.2 mg/kg/day compound dissolved in 250 mlof 10% glucose solution or in 250 ml of 0.9% NaCl solution, or byintravenous injection: 0.1-0.2 mg/kg/day compound dissolved in 10-20 mlof 10% glucose solution or of 0.9% NaCl solution, or by intraperitonealinjection (I.P.): 2.5 mg/kg/day compound dissolved in 10% glucosesolution or of 0.9% NaCl solution, or by oral administration wherein thedosage in mammal is 1-10 mg/Kg, 10-30 mg/Kg, 30-60 mg/Kg, or 60-90 mg/Kgcompound, or by intravenous injection or intravenous drip wherein thedosage in mammal is 0.01-0.1 mg/Kg, 0.1-0.2 mg/Kg, 0.2-0.4 mg/Kg, or0.4-0.6 mg/Kg compound, or by intraperitoneal injection (I.P.) whereinthe dosage in mammal is 1-3 mg/Kg, 3-5 mg/Kg, 4-6 mg/Kg, or 6-10 mg/Kgcompound.

The methods and uses of an isolated, modified or synthesized compound orits salt, ester, metabolite or derivative thereof, having the formulaof:

also named (1A),wherein R1 is selected from hydrogen, hydroxyl, O-angeloyl, O-tigloyl,O-senecioyl, O-alkyl, O-dibenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl,O-benzoyl alkyl substituted O-alkanoyl, O-alkanoyl substituted phenyl,O-alkenoyl substituted phenyl, O-aryl, O-acyl, O-heterocylic,O-heteroraryl, and derivatives thereof;R2 is selected from hydrogen, hydroxyl, O-angeloyl, O-tigloyl,O-senecioyl, O-alkyl, O-dibenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl,O-benzoyl alkyl substituted O-alkanoyl, O-alkanoyl substituted phenyl,O-alkenoyl substituted phenyl, O-aryl, O-acyl, O-heterocylic,O-heteroraryl, and derivatives thereof;R4 represents CH₂R6 or COR6, wherein R6 is selected from a groupconsisting of hydroxyl, O-angeloyl, O-tigloyl, O-senecioyl, O-alkyl,O-dibenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzoyl alkylsubstituted O-alkanoyl, O-alkanoyl substituted phenyl, O-alkenoylsubstituted phenyl, O-aryl, O-acyl, O-heterocylic, O-heteroraryl, andderivatives thereof; R3 is H or OH; R8 is H or OH;R5 is a hydrogen, heterocyclic or sugar moiety(ies), wherein the sugarmoiety(ies) is/are selected from a group consisting of glucose,galactose, rhamnose, arabinose, xylose, fucose, allose, altrose, gulose,idose, lyxose, mannose, psicose, ribose, sorbose, tagatose, talose,fructose, alduronic acid, glucuronic acid, galacturonic acid, andderivatives or combination thereof; wherein R9, R10, R11, R12, R13, R14,R15 are independently attached a group selecting from CH₃, CH₂OH, CHO,COOH, COO-alkyl, COO-aryl, COO-heterocyclic, COO-heteroaryl, CH₂Oaryl,CH₂O-heterocyclic, CH₂O-heteroaryl, alkyls group, hydroxyl, acetylgroup; wherein at least two of R1, R2 and R6 are comprising a groupselected from O-angeloyl, O-tigloyl, O-senecioyl, O-dibenzoyl,O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzoyl alkyl substitutedO-alkanoyl, O-alkanoyl substituted phenyl, O-alkenoyl substitutedphenyl, O-aryl, O-acyl, O-heterocylic, O-heteroraryl, and derivativesthereof; or at least one of R1, R2, and R4 is a sugar moiety substitutedwith at least two groups selected from a group consisting of angeloyl,acetyl, tigloyl, senecioyl, benzoyl, dibenzoyl, alkanoyl, alkenoyl,benzoyl alkyl substituted alkanoyl, O-alkanoyl substituted phenyl,O-alkenoyl substituted phenyl, aryl, acyl, heterocylic, heteroraryl, anda derivative thereof; or wherein R4 is CH₂R6; wherein R1 and R2independently consists an O-angeloyl group, or at least two of R1, R2and R6 are O-angeloyl or at least one of R1, R2 or R6 is a sugar moietywith two O-angeloyls; or wherein R5 is/are the sugar moiety(ies)selected from the following sugars and alduronic acids: glucose,galactose, rhamnose, arabinose, xylose, fucose, allose, altrose, gulose,idose, lyxose, mannose, psicose, ribose, sorbose, tagatose, talose,fructose, glucuronic acid, galacturonic acid; or their derivativesthereof, or the combination thereof; wherein the sugar comprisesglucuronic acid, arabinose and galactose. In an embodiment, wherein R5is/are sugar moiety(ies) selected from a group consisting of glucose,galactose, arabinose, alduronic acid, glucuronic acid, galacturonicacid, and a derivative or combination thereof; in embodiment, the acylhas 2 to 10 carbons. In an embodiment the method is administeringcontacting the compounds, wherein the compound is selected from thefollowing: a) An isolated, purified, modified or synthesized compoundhaving structure Xanifolia(Y),

or chemical name: 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosy(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β, 15α, 16α, 21β, 22α,28-hexahydroxyolean-12-ene;b) An isolated, purified, modified or synthesized compound havingstructure Xanifolia (Y1),

or chemical name: 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β,16α,21β, 22α, 28-pentahydroxyolean-12-ene; c) An isolated, purified,modified or synthesized compound having structure Xanifolia (Y2),

or chemical name: 3-O-[β-D-glucopyranosyl-(1→2)]-α-L-arabinofuranosy(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β, 15α, 16α, 21β, 22α,24β, 28-heptahydroxyolean-12-ene;d) An isolated, purified, modified or synthesized compound havingstructure Xanifolia (Y8),

or chemical name: 3-O-[β-glucopyranosyl (1→2)]-α-arabinofuranosyl (1→3)glucuronopyranosyl-21, 22-O-diangeloyl-3β, 16α, 21β, 22α, 24β,28-hexahydroxyolean-12-ene;e) An isolated, purified, modified or synthesized compound havingstructure Xanifolia (Y9),

or chemical name: 3-O-[β-galactopyranosyl(1→2)]-α-arabinofuranosyl(1→3)-β-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-rhamnopyranosyl-28-O-acetyl-3β,16α, 21β, 22α, 28-pentahydroxyolean-12-ene; and f) An isolated,purified, modified or synthesized compound having structure Xanifolia(Y10),

or chemical name:3-O-[β-galactopyranosyl(1→2)]-α-arabinofuranosyl(1→3)-β-glucuronopyranosyl-21, 22-O-diangeloyl-3β, 16α, 21β, 22α,28-pentahydroxyolean-12-ene;g) An isolated, purified, modified or synthesized compound havingstructure Xanifolia (Y0),

or chemical name: 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-angeloyl, 22-O-(2-methylpropanoyl)-3β,15α, 16α, 21β, 22α, 28-hexahydroxyolean-12-ene;h) An isolated, purified, modified or synthesized compound havingstructure Xanifolia (X),

or chemical name: 3-O-{[β-D-galactopyranosyl(1→2)]-[α-L-arabinofuranosyl(1→3)]-β-D-glucuronopyranoside butylester}-21-O-acetyl-22-O-angeloyl-3β,16α,21,22α,28-pentahydroxyolean-12-ene;i) An isolated, purified, modified or synthesized compound havingstructure (Y7),

or chemical name:3-O-[β-D-galactopyranosyl-(1→2)]-α-L-arabinofuranosyl-(1→3)-β-D-glucuronopyranosyl-21-O-angeloyl-28-O-2-methylbutanoyl-3β,15 α, 16α, 21β, 22α, 28-hexahydroxyolean-12-ene;j) An isolated, purified, modified or synthesized compound havingstructure (ACH):

In an embodiment the method is administering contacting the compound,wherein the compound is selected from the following:

k) An isolated, purified, modified or synthesized compound having astructure:

l) An isolated, purified, modified or synthesized compound having astructure (Y5):

In an embodiment, the method comprises administering to a subject orcontacting the cell with the compound, wherein the compound is isolated,purified, modified or synthesized having a structure selected fromfollowing formulae:

wherein R1, R2 are individually selected of an O-acetyl or O-angeloyl;wherein the R3, R4, R5, R6, R7 is hydrogen or hydroxyl. In anembodiment, the method comprises administering to a subject orcontacting the cell with compound in this application comprisingXanifolia Y0, Y1, Y2, Y, Y5, Y7, Y8, Y9, Y10, Xanifolia (x), Escin orAescin or a salt, ester, metabolite thereof. In an embodiment thecompounds may be selected from formulas (1A), (1B), (1C), (1D), (1E),(1F), (1G), (1H), (1J), (1K), (1L). In an embodiment, the compoundcomprises a triterpene backbone, two angeloyl groups and sugar moiety.In an embodiment the compound(s) are selected from Compound A to X andA1 to X1 in the application. In an embodiment the compound(s) areselected from Compound Z1 to Z13 in the application. In an embodimentthe method comprises administering to a subject or contacting the cellwith compound comprising of a triterpene wherein the carbon position 21,21 has an unsaturated group and sugar moieties at carbon 3.

In an embodiment, methods and compounds of this application reduce theability of bacteria, in colonization and tropism of cells. In anembodiment, methods and compounds of this application reduce theadhesive ability of cells or viruses in order to inhibit viruses bindingto host cells, wherein the viruses comprise HIV.

This invention provides a use of compound for manufacture of medicament,a method and a composition for modulating adhesion or anti-angiogenesisof cancer tumor, antiparasitics or manufacturing an adjuvantcomposition, wherein the modulating adhesion of cancer cell comprisingmodulating the secretion or expression of adhesion protein, wherein theadhesion proteins comprise fibronectin, integrins family, myosin,vitronectin, collagen, laminin, glycosylation cell surface proteins,polyglycans, cadherin, heparin, tenascin, CD 54, CAM, elastin and FAK;wherein the modulating comprises reducing, inhibiting and stimulating,wherein modulating adhesion protein comprises reducing the fibronectinfor inhibiting the metastasis or growth of cancer cells, wherein thecancer is selected from breast cancer, leukocytic cancer, liver cancer,ovarian cancer, bladder cancer, prostatic cancer, skin cancer, bonecancer, brain cancer, leukemia cancer, lung cancer, colon cancer, CNScancer, melanoma cancer, renal cancer, cervical cancer, esophagealcancer, testicular cancer, spleenic cancer, kidney cancer, lymphaticcancer, pancreatic cancer, stomach cancer and thyroid cancer; whereinmodulating adhesion of cancer cells comprises modulating the secretionor expression of adhesion protein, wherein the adhesion proteinscomprise fibronectin, integrins family, myosin, vitronectin, collagen,laminin, glycosylation cell surface proteins, polyglycans, cadherin,heparin, tenascin, CD 54, CAM, elastin and FAK; wherein modulatingcomprises reducing, inhibiting and stimulating; wherein modulatingangiogenesis comprises inhibiting and stimulating angiopoietin, whereincomprising angiopoietin 1, angiopoietin 2, angiopoietin 3, angiopoietin4, angiopoietin 5, angiopoietin 6 and angiopoietin 7; wherein theangiopoietin comprising angiopoietin-like 1, angiopoietin-like 2,angiopoietin-like 3, angiopoietin-like 4, angiopoietin-like 5,angiopoietin-like 6 and angiopoietin-like 7; wherein the modulatingcomprises positive and negative regulating; wherein modulatingangiopoietin comprises stimulating angiopoietin 2 in order to inhibitangiogenesis; wherein modulating angiopoietin comprises inhibitingangiopoietin 1 in order to inhibit angiogenesis; wherein modulatingangiopoietin comprises inhibiting angiopoietin-like 1; whereinmodulating angiopoietin comprises inhibiting angiopoietin-like 4;wherein the antiparasitics comprise inhibiting leishmaniases,amoebiasis, trypanosomiasis, toxoplasmosis or malaria, wherein themethod comprises administering to a subject or contacting the cell withthe compound in this application comprising Xanifolia Y0, Y1, Y2, Y, Y5,Y7, Y8, Y9, Y10, Xanifolia (x), Escin or Aescin or a salt, ester,metabolite thereof. In an embodiment the compound may be selected fromformulas (1A), (1B), (1C), (1D), (1E), (1F), (1G), (1H), (1J), (1K),(1L), or a salt, ester, acid, metabolite thereof. In an embodiment, thecompound comprises a triterpene backbone, two angeloyl groups and sugarmoiety. In an embodiment the compound(s) are selected from Compound A toX and A1 to X1, or a salt, ester, acid, metabolite thereof in theapplication. In an embodiment the compound(s) are selected from CompoundZ1 to Z13 in the application. In an embodiment the compound(s) areselected from ACH-Z4, ACH-Y10, ACH-Y2, ACH-Y8, ACH-Y7, ACH-Y0, ACH-X,ACH-E, ACH-Mb5 and ACHMb12, or a salt, ester, acid, metabolite thereof.In an embodiment the saponins comprise Ba1, Ba2, Ba3, Ba4, Ba5, Ba6,Ba7, Ba8, Ba9, Ba10, Ba11, Ba12, Ba13, Ba14, Ba15, Ba16, and Ba17, or asalt, ester, acid, metabolite thereof. In an embodiment the saponinscomprise Mb1, Mb2, Mb3, Mb4, Mb5, Mb6, Mb7, Mb8, Mb9, Mb10, Mb11, Mb12,and Mb13, or a salt, ester, acid, metabolite thereof.

In an embodiment, the methods and compositions of this application canbe used for manufacturing an adjuvant vaccine, wherein the methods andcompositions are used for manufacturing an adjuvant vaccine in asubject, wherein the method comprises administering to a subject orcontacting the cell with the compound in this application comprisingXanifolia Y0, Y1, Y2, Y, Y5, Y7, Y8, Y9, Y10, Xanifolia (x), Escin orAescin or a salt, ester, metabolite thereof. In an embodiment thecompound may be selected from formulas (1A), (1B), (1C), (1D), (1E),(1F), (1G), (1H), (1J), (1K), (1L), or a salt, ester, acid, metabolitethereof. In an embodiment, the compound comprises a triterpene backbone,two angeloyl groups and sugar moiety. In an embodiment the compound(s)are selected from Compound A to X and A1 to X1, or a salt, ester, acid,metabolite thereof in the application. In an embodiment the compound(s)are selected from Compound Z1 to Z13 in the application. In anembodiment the compound(s) are selected from ACH-Z4, ACH-Y10, ACH-Y2,ACH-Y8, ACH-Y7, ACH-Y0, ACH-X, ACH-E, ACH-Mb5 and ACH-Mb12, or a salt,ester, acid, metabolite thereof. In an embodiment the saponins compriseBa1, Ba2, Ba3, Ba4, Ba5, Ba6, Ba7, Ba8, Ba9, Ba10, Ba11, Ba12, Ba13,Ba14, Ba15, Ba16, and Ba17, or a salt, ester, acid, metabolite thereof.In an embodiment the saponins comprise Mb1, Mb2, Mb3, Mb4, Mb5, Mb6,Mb7, Mb8, Mb9, Mb10, Mb11, Mb12 and Mb13, or a salt, ester, acid,metabolite thereof. In an embodiment, this application provides anadjuvant composition comprising a saponin or compound selecting fromabove; wherein the composition is comprised of an immunostimulatoryoligonucleotide.

In an embodiment, the methods and compositions of this application canbe used for manufacture of medicament for vaccine or antiviral agent forEnterovirus comprising EV71, wherein the method comprises administeringto a subject or contacting the cell with compounds selected in thisapplication comprising Mb1, Mb2, Mb3, Mb4, Mb5, Mb6, Mb7, Mb8, Mb9,Mb10, Mb11, Mb12 and Mb13, or a salt, ester, acid, metabolite thereof.

The composition comprises bioactive compounds from natural plants orsynthesis.

The majority of the plants are from the Sapindaceae family, which has140-150 genera with 1400-2000 species. The program is based on ourpurification methods and biological assays including the MTT assay SeeInternational Application No. PCT/US05/31900, filed Sep. 7, 2005, U.S.Ser. No. 11/289,142, filed Nov. 28, 2005, and U.S. Ser. No. 11/131,551,filed May 17, 2005, and PCT/US2008/002086, 1188-ALA-PCT, filed Feb. 15,2008, the contents of which are incorporated herein by reference. Thedetails of Analysis of gene expression of ES2 cells after Y-treatment byMicroarray, Data Analysis Methods and Western blot in PCT/US2008/002086,1188-ALA-PCT, filed Feb. 15, 2008, the contents of which areincorporated herein by reference.

This invention provides a composition comprising an effective amount oftriterpenoidal saponins named as Xanifolia Y1, Y2, Y, Y5, Y7, Y8, Y9,Y10, and Y0 or their salt or their derivatives. In an embodiment thesaponins comprise Ba1, Ba2, Ba3, Ba4, Ba5, Ba6, Ba7, Ba8, Ba9, Ba10,Ba11, Ba12, Ba13, Ba14, Ba15, Ba16, and Ba17 or their salt or theirderivatives. In an embodiment the saponins comprise Mb1, Mb2, Mb3, Mb4,Mb5, Mb6, Mb7, Mb8, Mb9, Mb10, Mb11, Mb12 and Mb13, or their salt ortheir derivatives for modulating expression or secretion of adhesionproteins, reducing expression or secretion of adhesion proteins orreducing the expression or secretion of fibronectin, for treatingchronic venous insufficiency, peripheral edema, antilipemic, chronicvenous disease, varicose vein disease, varicose syndrome, venous stasis,expectorant, peripheral vascular disorders, cerebro-organic convulsion,cerebral circulation disorder, cerebral edema, psychoses, dysmenorrheal,hemorrhoids, episiotomies, peripheral edema formation or postoperativeswelling; for reducing symptoms of pain; for reducing symptoms ofstomach pain; for reducing symptoms of leg pain; for treating pruritis,lower leg volume, thrombosis, thromophlebitis; for treating rheumatism;for preventing gastric ulcers antispasmotic; blocking the migration,metastasis of cancer cells or inhibiting tumor growth. In an embodimentthe method comprises administering to a subject or contacting the cellwith compounds in this application comprising Xanifolia Y0, Y1, Y2, Y,Y5, Y7, Y8, Y9, Y10, Xanifolia (x), Escin or Aescin or a salt, ester,metabolite thereof. In an embodiment the compound may be selected fromformulas (1A), (1B), (1C), (1D), (1E), (1F), (1G), (1H), (1J), (1K),(1L), or a salt, ester, acid, metabolite thereof. In an embodiment, thecompound comprises a triterpene backbone, two angeloyl groups and sugarmoiety. In an embodiment the compound(s) are selected from Compound A toX and A1 to X1 in the application. In an embodiment the compound(s) areselected from Compound Z1 to Z13 in the application. In an embodimentthe compound(s) are selected from ACH-Z4, ACH-Y10, ACH-Y2, ACH-Y8,ACH-Y7, ACH-Y0, ACH-X, ACH-E, ACH-Mb5 and ACH-Mb12, or a salt, ester,acid, metabolite thereof. The compounds of this invention can beisolated from natural sources or synthesized.

See experiments results in this application and see PCT/US05/31900,filed Sep. 7, 2006; U.S. Ser. No. 10/906,303, filed Feb. 14, 2005;International Application No. PCT/US04/43465, filed Dec. 23, 2004;International Application No. PCT/US04/33359, filed Oct. 8, 2004 andU.S. Ser. No. 11/131,551, filed May 17, 2005, PCT/US2007/077273, filedAug. 30, 2007, PCT/US2008/002086, 1188-ALA-PCT, filed Feb. 15, 2008,U.S. 61/038,277 filed Mar. 20, 2008, U.S. 61/054,308, filed May 19,2008, the contents of which are incorporated herein by reference.

Acid Hydrolysis of Saponin

15 mg Xanifolia-Y was dissolved in 1 ml of Methanol. 1 ml of 2N HCl wasthen added. The mixture was refluxed in 80 C water bath for 5 hours. Thesolution was then neutralized by adding 2 ml of 1N NaOH (to final pH4-6). The aglycone was then extracted with ethylacetate 3 ml×2. Theextracts were collected and pooled.

Further isolation of aglycone (ACH-Y) was achieved by HPLC withisocratic elution of 80-100% acetonitrile. Repeat the experiment withcompound Z4, Y10, Y2, Y8, Y7, Y0, X, and ESCIN were obtained compoundsACH-Z4, ACH-Y10, ACH-Y2, ACH-Y8, ACH-Y7, ACH-Y0, ACH-X, ACH-E, ACH-Mb12,ACH-Mb5. In mild conditions, the saponin will be partially hydrolyzed toa mixture of products. The products can be separated by HPLC. Also,specific partial hydrolysis can be achieved with enzymes. Theβ-glucosidase is a good enzyme for cleaving the β-glucose from saponins.

Removal of the acyl group by alkaline hydrolysis

20 mg of Xanifolia-Y was dissolved in 0.5 ml of 1M NaOH. The solutionwas incubated in 80 C water bath for 4 hours. It was cooled to roomtemperature before neutralized with 0.5 ml 1 N HCl (adjust pH to about3). The mixture was extracted with 2 ml 1-butanol 3 times. The butanolfractions were collected and lyophilized. The hydrolyzed saponin withfurther purified with HPLC in a C-18 column eluted with 25%acetonitrile.

Compounds AKOH-Y and AKOH-Mb5 have lost anticancer activity. Thisinvention provides a use of compound for manufacture of medicament or amethod of modulating adhesion proteins or their receptors, reducing theadhesive ability of the cancer cells, wherein the modulating comprisespositive or negative regulating. In an embodiment, the adhesion proteinscomprise fibronectin, integrins family, myosin, vitronectin, collagen,laminin, glycosylation cell surface proteins, polyglycans, cadherin,heparin, tenascin, CD 54, CAM, elastin and FAK. In an embodiment, themethod comprises reducing the secretion of fibronectin. This inventionprovides a method of blocking the migration, metastasis of cancer cellsor inhibiting cancer cell growth or inhibiting leishmaniases modulatingadhesion or inhibiting angiogenesis of cancer tumor, antiparasitics ormanufacturing an adjuvant composition comprising administering aneffective amount of a pharmaceutical composition comprising acomposition comprises the molecular formula or compound in thisinvention. The cancers comprise breast cancer, leukocytic cancer, livercancer, ovarian cancer, bladder cancer, prostatic cancer, skin cancer,bone cancer, brain cancer, leukemia cancer, lung cancer, colon cancer,CNS cancer, melanoma cancer, renal cancer, cervical cancer, esophagealcancer, testicular cancer, spleenic cancer, kidney cancer, lymphaticcancer, pancreatic cancer, stomach cancer and thyroid cancer. Thecompounds of this invention can be isolated from natural sources orsynthesized. In an embodiment the method comprises administering to asubject or contacting a cell with the compounds, wherein the compound isselected from the following:

(Z1)3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-angeloyl,22-O-(2-methylpropanoyl)-3β, 15α, 16α, 21β, 22α,28-hexahydroxyolean-12-ene;

(Z2)3-O-[β-D-galactopyranosyl-(1→2)]-α-L-arabinofuranosyl-(1→3)-β-D-glucuronopyranosyl-21-O-angeloyl-22-O-(angeloyl-2-methylbutanoyl)-3β,15 α, 16α, 21β, 22α, 28-hexahydroxyolean-12-ene;

(Z3)3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(2-methylpropanoyl),22-O-(2-methylpropanoyl)-3β, 15α, 16α, 21β, 22α,28-hexahydroxyolean-12-ene;

(Z4)3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-angeloyl,22-O-benzoyl-3β, 15α, 16α, 21β, 22α, 28-hexahydroxyolean-12-ene;

(Z5) 3-O-[β-D-galaotopyranosyl (1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-angeloyl, 22-O-angeloyl-3β, 15α, 16α,21β, 22α, 28-hexahydroxyolean-12-ene;

(Z6) 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(2-methylpropanoyl)-O-benzoyl,22-O-(2-methylpropanoyl)-3β, 15α, 16α, 21β, 22α,28-hexahydroxyolean-12-ene;

(Z7) 3-O-[β-D-galaotopyranosyl (1→2)]-α-L-arabinofuranosyl(1→3)-β-D-gluouronopyranosyl-21-O-(2-methylpropanoyl)-O-angeloyl,22-O-(2-methylbutanoyl)-3β, 15α, 16α, 21β, 22α,28-hexahydroxyolean-12-ene;

(Z8) 3-O-[β-D-galaotopyranosyl (1→2)]-α-L-arabinofuranosyl(1→3)-β-D-gluouronopyranosyl-21-O-benzoyl, 22-O-benzoyl-3β, 15α, 16α,21β, 22α, 28-hexahydroxyolean-12-ene;

(Z9) 3-O-[β-D-galactopyranosyl(1→2)]-β-D-xylopyranosyl(1→3)-β-D-glucuronopyranosyl-21-O-angeloyl, 22-O-benzoyl-3β, 15α, 16α,21β, 22α, 28-hexahydroxyolean-12-ene;

3-O-[β-D-galactopyranosyl(1→2)]-β-D-xylopyranosyl-(1→3)-β-D-glucuronopyranosyl-21-O-(2-methylpropanoyl),22-O-(2-methylpropanoyl)-3β, 15α, 16α, 21β, 22α,28-hexahydroxyolean-12-ene;

3-O-[β-D-galactopyranosyl-(1→2)]-β-D-xylopyranosyl-(1→3)-β-D-glucuronopyranosyl-21-O-angeloyl-22-O-(angeloyl-2-methylbutanoyl)-3β,15α, 16α, 21β, 22α, 28-hexahydroxyolean-12-ene;

3-O-[β-D-galactopyranosyl(1→2)]-β-D-xylopyranosyl(1→3)-β-D-glucuronopyranosyl-21-O-angeloyl, 22-O-tigloyl-3β, 16α, 21β,22α, 28-pentahydroxyolean-12-ene;

3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-angeloyl,22-O-tigloyl-3β, 15α, 16α, 21β, 22α, 28-hexahydroxyolean-12-ene.

This invention provides uses of a compound for manufacture of medicamentselected from formula (1B), for modulating, regulating or interactingwith the adhesion protein, wherein the adhesion proteins comprisefibronectin, integrins family, myosin, vitronectin, collagen, laminin,glycosylation cell surface proteins, polyglycans, cadherin, heparin,tenascin, CD 54, CAM, elastin and FAK. In an embodiment, this inventionprovides a method of reducing the expression or secretion offibronectin, modulating adhesion or angiogenesis of cancer cells,antiparasitics, enhancing an immune response, providing adjuvantactivities or providing vaccine activities, inhibiting cancer metastasisor growth, using the compounds selected for the following:

also named as (1B),or a salt, ester, metabolite or derivative thereof, wherein R1 comprisesa group selected from hydrogen, angeloyl, acetyl, tigloyl, senecioyl,alkyl, dibenzoyl, benzoyl, alkanoyl, alkenoyl, benzoyl alkyl substitutedalkanoyl, acyl, aryl, heterocylic, heteroraryl, alkenylcarbonyl andderivatives thereof; R2 comprises a group selected from hydrogen,angeloyl, acetyl, tigloyl, senecioyl, alkyl, benzoyl, dibenzoyl,alkanoyl, alkenoyl, benzoyl alkyl substituted alkanoyl, aryl, acyl,heterocylic, heteroraryl, alkenylcarbonyl and derivative thereof; R4represents CH₂OR6 or COOR6, wherein R6 is selected from hydrogen,angeloyl, acetyl, tigloyl, senecioyl, alkyl, benzoyl, dibenzoyl,alkanoyl, alkenoyl, benzoyl alkyl substituted alkanoyl, aryl, acyl,heterocylic, heteroraryl and derivative thereof; R3 is H or OH; whereinat least one of R1, R2, and R6 comprises a group selected from angeloyl,acetyl, tigloyl, senecioyl, benzoyl, dibenzoyl, alkanoyl, alkenoyl,benzoyl alkyl substituted alkanoyl, aryl, acyl, heterocylic, heteroraryland derivative thereof; R5 comprises a hydrogen or sugar moiety, whereinthe sugar moiety comprises at least one sugar of, but is not limited to,D-glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, alduronicacid: D-glucuronic acid, D-galacturonic acid or a derivative thereof, orthe combination thereof. In an embodiment, R1 comprises a sugar moietywherein substituted with two groups selecting from angeloyl, acetyl,tigloyl, senecioyl, benzoyl, dibenzoyl, alkanoyl, alkenoyl, benzoylalkyl substituted alkanoyl, aryl, acyl, heterocylic heteroraryl and aderivative thereof. In an embodiment, R1 comprises a sugar moietywherein substituted with at least one group selecting from angeloyl,acetyl, tigloyl, senecioyl, benzoyl, dibenzoyl, alkanoyl, alkenoyl,benzoyl alkyl substituted alkanoyl, aryl, acyl, heterocylic, heteroraryland a derivative thereof. In an embodiment, R2 comprises a sugar moietywherein at least one group is selected from angeloyl, acetyl, tigloyl,senecioyl, alkyl, benzoyl, dibenzoyl, alkanoyl, alkenoyl, benzoyl alkylsubstituted alkanoyl, aryl, acyl, heterocylic, heteroraryl and aderivative thereof. In an embodiment, R2 comprises a sugar moiety or aside chain wherein at least two groups are selected from angeloyl,acetyl, tigloyl, senecioyl, alkyl, benzoyl, dibenzoyl, alkanoyl,alkenoyl, benzoyl alkyl substituted alkanoyl, aryl, acyl, heterocylic,heteroraryl and a derivative thereof. In an embodiment, R4 comprisesCH₂OR6 or COOR6 wherein R6 is a sugar moiety which comprises at leastone group selected from angeloyl, acetyl, tigloyl, senecioyl, benzoyl,dibenzoyl, alkanoyl, alkenoyl, benzoyl alkyl substituted alkanoyl, aryl,acyl, heterocylic, heteroraryl and a derivative thereof.

In an embodiment, R4 comprises CH₂OR6 or COOR6, wherein R6 is a sugarmoiety which comprises at least two groups selected from angeloyl,acetyl, tigloyl, senecioyl, benzoyl, dibenzoyl, alkanoyl, alkenoyl,benzoyl alkyl substituted alkanoyl, aryl, acyl, heterocylic, heteroraryland a derivative thereof. In an embodiment, R4 comprises CH₂OR6 orCOOR6, wherein R6 is a sugar moiety which comprises at least two groupsselected from angeloyl, acetyl, tigloyl and senecioyl. In an embodiment,R4 comprises CH₂OR6 or COOR6 of formula (1B), at least two of R1, R2 andR6 comprise the group selected from angeloyl, acetyl, tigloyl,senecioyl, benzoyl, dibenzoyl, alkanoyl, alkenoyl, benzoyl alkylsubstituted alkanoyl, aryl, acyl, heterocylic, heteroraryl and aderivative thereof. In an embodiment, R4 comprises CH₂OR6 or COOR6 offormula (1B), wherein at least two of R1, R2 and R6 comprise angeloyl,benzoyl, alkenoyl, or a derivative thereof. In an embodiment, R4 is aside chain comprising CH₂OCOCH₃, CH₂COO-alkyl, CH₂OH, COOH, angeloyl,acetyl, tigloyl, senecioyl, alkyl, benzoyl, dibenzoyl, alkanoyl,alkenoyl, benzoyl alkyl substituted alkanoyl, aryl, acyl, heterocylic orheteroraryl, alkanoyl substituted phenyl, alkenoyl substituted phenyl,or a derivative thereof. In a further embodiment, R5 comprises a sugarmoiety, wherein the sugar moiety comprises one or more sugar of, but isnot limited to glucose, galactose, rhamnose, arabinose, xylose, fucose,allose, altrose, gulose, idose, lyxose, mannose, psicose, ribose,sorbose, tagatose, talose, fructose, or alduronic acid: glucuronic acid,galacturonic acid, or derivatives thereof, or the combination thereof.In an embodiment, R5 comprises a sugar moiety or a group capable ofperforming the function of the sugar moiety. In an embodiment, the R5represents H. In an embodiment, R4 represents H, OH or CH₃. In anembodiment, positions C23, C24, C25, C26, C29 and C30 of the compoundindependently comprise CH₃, CH₂OH, CHO, COOH, COOa-lkyl, COO-aryl,COO-heterocyclic, COO-heteroaryl, CH₂Oaryl, CH₂O-heterocyclic,CH₂O-heteroaryl, alkyls group, acetyl group or derivatives thereof. Inan embodiment, R1 and R2 independently comprise an angeloyl group. In anembodiment, R1 is a sugar moiety or a side chain which comprises twoangeloyl groups. In an embodiment, R1 and R2 independently comprise abenzoyl group. In an embodiment, R1 is a sugar moiety which issubstituted with two benzoyl groups. In an embodiment, R₃ represents Hor OH. In an embodiment, R8 may be OH. In an embodiment, the O at C21,22 may be replaced by NH. In an embodiment, this invention provides amethod of reducing the secretion of fibronectin; wherein the medicamentis for inhibiting tumor or cancer cell growth and for treating cancer,wherein the cancers comprise breast cancer, leukocytic cancer, livercancer, ovarian cancer, bladder cancer, prostatic cancer, skin cancer,bone cancer, brain cancer, leukemia cancer, lung cancer, colon cancer,CNS cancer, melanoma cancer, renal cancer, cervical cancer, esophagealcancer, testicular cancer, spleenic cancer, kidney cancer, lymphaticcancer, pancreatic cancer, stomach cancer and thyroid cancer.Substitution, deletion and/or addition of any group in theabove-described compounds by other group(s) will be apparent to one ofordinary skill in the art based on the teachings of this application. Ina further embodiment, the substitution, deletion and/or addition of thegroup(s) in the compound of the invention does not substantially affectthe biological function of the compound.

This invention provides uses, methods, processes, compounds andcompositions for modulating adhesion or angiogenesis of cancer cells,antiparasitics, enhancing an immune response, providing adjuvantactivities or providing vaccine activities, inhibiting cancer metastasisor growth, reducing adhesion protein of cells, wherein the adhesionproteins comprise fibronectin, integrins family, myosin, vitronectin,collagen, laminin, glycosylation cell surface proteins, polyglycans,cadherin, heparin, tenascin, CD 54, CAM, elastin and FAK. In anembodiment, methods comprise inhibiting the gene expression. In anembodiment, this invention provides a method of reducing the expressionor secretion of fibronectin. In an embodiment the method can block themigration, metastasis of cancer cells or inhibit the growth of cancersor anti-angiogenesis, wherein the cancers comprise breast cancer,leukocytic cancer, liver cancer, ovarian cancer, bladder cancer,prostate cancer, skin cancer, bone cancer, brain cancer, leukemiacancer, lung cancer, colon cancer, CNS cancer, melanoma cancer, renalcancer, cervical cancer, esophageal cancer, testicular cancer, spleeniccancer, kidney cancer, lymphatic cancer, pancreatic cancer, stomachcancer and thyroid cancer, In an embodiment the compounds areanti-angiogenic, inhibit cancer cell metastasis and inhibit cancergrowth. In an embodiment the compounds promote angiopoietin 2. In anembodiment the compound is selected from the following formulas (1E). Inan embodiment the method comprises administering to a subject orcontacting the cells with the compounds, wherein the compound isselected from the formula (1E):

also named (1E), whereinR1 is selected from hydrogen, hydroxyl, O-angeloyl, O-tigloyl,O-senecioyl, O-alkyl, O-dibenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl,O-benzoyl alkyl substituted O-alkanoyl, O-alkanoyl substituted phenyl,O-alkenoyl substituted phenyl, O-aryl, O-acyl, O-heterocylic,O-heteroraryl, O-alkenylcarbonyl and derivatives thereof;R2 is selected from hydrogen, hydroxyl, O-angeloyl, O-tigloyl,O-senecioyl, O-alkyl, O-dibenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl,O-benzoyl alkyl substituted O-alkanoyl, O-alkanoyl substituted phenyl,O-alkenoyl substituted phenyl, O-aryl, O-acyl, O-heterocylic,O-heteroraryl, O-alkenylcarbonyl and derivatives thereof;R4 represents CH3, CHO, CH₂R6 or COR6, wherein R6 is selected fromhydroxyl, O-angeloyl, O-tigloyl, O-senecioyl, O-alkyl, O-dibenzoyl,O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzoyl alkyl substitutedO-alkanoyl, O-alkanoyl substituted phenyl, O-alkenoyl substitutedphenyl, O-aryl, O-acyl, O-heterocylic, O-heteroraryl, O-alkenylcarbonyland derivatives thereof; R3 is H or OH; R8 is H or OH, particularly OH;R16 is H, or R4 and R16 may together form —CH2-X—, CH(OH)—X— orC(═O)—X—, wherein the —X— may be 0 or NH; wherein when the C12-13 ofring 3 of the triterpene has a double bond then R16 is absent; R5 is ahydrogen, heterocyclic or sugar moiety(ies), wherein the sugarmoiety(ies) is/are selected from a group consisting of glucose,galactose, rhamnose, arabinose, xylose, fucose, allose, altrose, gulose,idose, lyxose, mannose, psicose, ribose, sorbose, tagatose, talose,fructose, alduronic acid, glucuronic acid, galacturonic acid, andderivatives or combination thereof; wherein R9, R10, R11, R12, R13, R14,R15 are independently attached a group selecting from CH₃, CH₂OH, CHO,COOH, COO-alkyl, COO-aryl, COO-heterocyclic, COO-heteroaryl, CH₂Oaryl,CH₂O-heterocyclic, CH₂O-heteroaryl, alkyls group, hydroxyl, acetylgroup; or wherein R9, R10, R11, R12, R13, R14, R15 are independentlyattached a CH₃; wherein R4 and R16 form a divalent radical of formulaCH2O, CH(OR7)O, or COOR7, wherein R7 is hydrogen, alkyl, angeloyl,tigloyl, senecioyl, dibenzoyl, benzoyl, alkanoyl, alkenoyl, benzoylalkyl substituted alkanoyl, aryl, acyl, heterocylic, heteroraryl, andderivatives thereof; wherein at least two of R1, R2 and R6 individuallycomprises a group selected from O-angeloyl, O-tigloyl, O-senecioyl,O-dibenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzoyl alkylsubstituted O-alkanoyl, O-aryl, O-acyl, O-heterocylic, O-heteroraryl,and derivatives thereof; or at least one of R1, R2, and R4 is a sugarmoiety substituted with at least two groups selected from a groupconsisting of angeloyl, acetyl, tigloyl, senecioyl, benzoyl, dibenzoyl,alkanoyl, alkenoyl, benzoyl alkyl substituted alkanoyl, aryl, acyl,heterocylic, heteroraryl, and a derivative thereof; or wherein R4 isCH₂R6; wherein R1 and R2 independently consists an O-angeloyl group, orat least two of R1, R2 and R6 are O-angeloyl or at least one of R1, R2or R6 is a sugar moiety with two O-angeloyls; wherein R5 is/are thesugar moiety(ies) selected from the following sugars and alduronicacids: glucose, galactose, rhamnose, arabinose, xylose, fucose, allose,altrose, gulose, idose, lyxose, mannose, psicose, ribose, sorbose,tagatose, talose, fructose, glucuronic acid, galacturonic acid; or theirderivatives thereof, or the combination thereof; wherein the sugarpreferably comprises glucuronic acid, arabinose and galactose. In anembodiment, wherein R5 is/are sugar moiety(ies) selected from a groupconsisting of glucose, galactose, arabinose, alduronic acid, glucuronicacid, galacturonic acid, and a derivative or combination thereof. In anembodiment, wherein R5 is3-β-O-{[(α-L-rhamnopyranosyl-(1→2)]-α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranosyl-(1→3)]-[β-D-galactopyranosyl-(1→2)]-β-D-glucuronopyranosyl}.In an embodiment, wherein the carbon ring 3 comprises a double bond whenR16 is H; wherein the double bond in carbon ring 3 is reduced when R4and R16 form a divalent radical. In an embodiment, the compound has nosugar moiety. In an embodiment, the compound has at least 1 sugarmoiety(ies). In an embodiment, the compound has at least 2 sugarmoiety(ies). In an embodiment, the compound has at least 3 sugarmoieties. In an embodiment, the compound has at least 4 sugar moieties.In an embodiment, the compound has at least 5 sugar moieties. In anembodiment, the number of sugar moiety(ies) at R5 is(are) 1, 2, 3, 4, or5. In an embodiment, the sugar moieties attach at R5 or other sidebonds. In an embodiment, the sugar moiety may be in the form ofalduronic acid. In an embodiment, the compound is attached an acid. Inan embodiment, the compound is in form of salts.

In an embodiment the method comprises administering to a subject orcontacting the cells with the compounds, wherein the compound isselected from the formula (1F):

also named (1F), whereinR1 is selected from hydrogen, hydroxyl, O-angeloyl, O-tigloyl,O-senecioyl, O-alkyl, O-dibenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl,O-benzoyl alkyl substituted O-alkanoyl, O-alkanoyl substituted phenyl,O-alkenoyl substituted phenyl, O-aryl, O-acyl, O-heterocylic,O-heteroraryl, O-alkenylcarbonyl and derivatives thereof;R2 is selected from hydrogen, hydroxyl, O-angeloyl, O-tigloyl,O-senecioyl, O-alkyl, O-dibenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl,O-benzoyl alkyl substituted O-alkanoyl, O-alkanoyl substituted phenyl,O-alkenoyl substituted phenyl, O-aryl, O-acyl, O-heterocylic,O-heteroraryl, O-alkenylcarbonyl and derivatives thereof;R4 is selected from hydrogen, hydroxyl, O-angeloyl, O-tigloyl,O-senecioyl, O-alkyl, O-dibenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl,O-benzoyl alkyl substituted O-alkanoyl, O-alkanoyl substituted phenyl,O-alkenoyl substituted phenyl, O-aryl, O-acyl, O-heterocylic,O-heteroraryl, O-alkenylcarbonyl and derivatives thereof; R3 is H or OH;R8 is H or OH, particularly OH;R5 is a hydrogen or sugar moiety(ies), wherein the sugar moiety(ies)is/are selected from a group consisting of glucose, galactose, rhamnose,arabinose, xylose, fucose, allose, altrose, gulose, idose, lyxose,mannose, psicose, ribose, sorbose, tagatose, talose, fructose, alduronicacid, glucuronic acid, galacturonic acid, and derivatives or combinationthereof; wherein R9, R10, R11, R12, R13, R14, R15 are independentlyattached a group selecting from CH₃, CH₂OH, CHO, COOH, COO-alkyl,COO-aryl, COO-heterocyclic, COO-heteroaryl, CH₂Oaryl, CH₂O-heterocyclic,CH₂O-heteroaryl, alkyls group, hydroxyl, acetyl group; or wherein R9,R10, R11, R12, R13, R14, R15 are independently attached a CH₃; whereinat least two of R1, R2 and R4 are comprising a group selected fromO-angeloyl, O-tigloyl, O-senecioyl, O-dibenzoyl, O-benzoyl, O-alkanoyl,O-alkenoyl, O-benzoyl alkyl substituted O-alkanoyl, O-aryl, O-acyl,O-heterocylic, O-heteroraryl, and derivatives thereof; or at least oneof R1, R2, and R4 is a sugar moiety substituted with at least two groupsselected from a group consisting of angeloyl, acetyl, tigloyl,senecioyl, benzoyl, dibenzoyl, alkanoyl, alkenoyl, benzoyl alkylsubstituted alkanoyl, aryl, acyl, heterocylic, heteroraryl, and aderivative thereof; or wherein R4, R1 and R2 independently consists anO-angeloyl group, or at least two of R1, R2 and R4 are O-angeloyl or atleast one of R1, R2 or R4 is a sugar moiety with two O-angeloyls;wherein R5 is/are the sugar moiety(ies) selected from the followingsugars and alduronic acids: glucose, galactose, rhamnose, arabinose,xylose, fucose, allose, altrose, gulose, idose, lyxose, mannose,psicose, ribose, sorbose, tagatose, talose, fructose, glucuronic acid,galacturonic acid; or their derivatives thereof, or the combinationthereof; wherein the sugar preferably comprises glucuronic acid,arabinose and galactose. In an embodiment, wherein R5 is/are sugarmoiety(ies) selected from a group consisting of glucose, galactose,arabinose, alduronic acid, glucuronic acid, galacturonic acid, and aderivative or combination thereof; In an embodiment, wherein R5 is3-β-O-{[(α-L-rhamnopyranosyl-(1→2)]-α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranosyl-(1→3)]-[β-D-galactopyranosyl-(1→2)]-β-D-glucuronopyranosyl}.In an embodiment, the compound is in form of salts.

In an embodiment the method comprises administering to a subject orcontacting the cells with the compounds of following:

also named (1G), whereinR1 is selected from hydrogen, hydroxyl, O-angeloyl, O-tigloyl,O-senecioyl, O-alkyl, O-dibenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl,O-benzoyl alkyl substituted O-alkanoyl, O-alkanoyl substituted phenyl,O-alkenoyl substituted phenyl, O-aryl, O-acyl, O-heterocylic,O-heteroraryl, O-alkenylcarbonyl and derivatives thereof;R2 is selected from hydrogen, hydroxyl, O-angeloyl, O-tigloyl,O-senecioyl, O-alkyl, O-dibenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl,O-benzoyl alkyl substituted O-alkanoyl, O-alkanoyl substituted phenyl,O-alkenoyl substituted phenyl, O-aryl, O-acyl, O-heterocylic,O-heteroraryl, O-alkenylcarbonyl and derivatives thereof;R4 represents CH3, CHO, CH₂R6 or COR6, wherein R6 is selected fromhydroxyl, O-angeloyl, O-tigloyl, O-senecioyl, O-alkyl, O-dibenzoyl,O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzoyl alkyl substitutedO-alkanoyl, O-alkanoyl substituted phenyl, O-alkenoyl substitutedphenyl, O-aryl, O-acyl, O-heterocylic, O-heteroraryl, O-alkenylcarbonyland derivatives thereof; R3 is H or OH; R5 is H or OH; wherein R6, R7,R8, R9, R10, R11, R12 are independently attached a group selecting fromCH₃, CH₂OH, CHO, COOH, COO-alkyl, COO-aryl, COO-heterocyclic,COO-heteroaryl, CH₂Oaryl, CH₂O-heterocyclic, CH₂O-heteroaryl, alkylsgroup, hydroxyl, acetyl group.

In an embodiment, the compound is in form of salts.

In an embodiment the method comprises administering to a subject orcontacting the cells with the compounds of following:

also named (1H), whereinR1 is selected from hydrogen, hydroxyl, O-angeloyl, O-tigloyl,O-senecioyl, O-alkyl, O-dibenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl,O-benzoyl alkyl substituted O-alkanoyl, O-alkanoyl substituted phenyl,O-alkenoyl substituted phenyl, O-aryl, O-acyl, O-heterocylic,O-heteroraryl, O-alkenylcarbonyl and derivatives thereof; R2 is selectedfrom hydrogen, hydroxyl, O-angeloyl, O-tigloyl, O-senecioyl, O-alkyl,O-dibenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzoyl alkylsubstituted O-alkanoyl, O-alkanoyl substituted phenyl, O-alkenoylsubstituted phenyl, O-aryl, O-acyl, O-heterocylic, O-heteroraryl,O-alkenylcarbonyl and derivatives thereof; R4 is selected from hydroxyl,CH2OH, O-angeloyl, O-tigloyl, O-senecioyl, O-alkyl, O-dibenzoyl,O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzoyl alkyl substitutedO-alkanoyl, O-alkanoyl substituted phenyl, O-alkenoyl substitutedphenyl, O-aryl, O-acyl, O-heterocylic, O-heteroraryl, O-alkenylcarbonyland derivatives thereof; R3 is H or OH; R5 is H or OH; wherein R6, R7,R8, R9, R10, R11, R12 are independently attached a group selecting fromCH₃, CH₂OH, CHO, COOH, COO-alkyl, COO-aryl, COO-heterocyclic,COO-heteroaryl, CH₂Oaryl, CH₂O-heterocyclic, CH₂O-heteroaryl, alkylsgroup, hydroxyl, acetyl group. In an embodiment, the compound is in formof salts.

In an embodiment the method comprises administering to a subject orcontacting the cells with the compounds of following:

also named (1J), whereinR1 is selected from hydrogen, hydroxyl, O-angeloyl, O-tigloyl,O-senecioyl, O-alkyl, O-dibenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl,O-benzoyl alkyl substituted O-alkanoyl, O-alkanoyl substituted phenyl,O-alkenoyl substituted phenyl, O-aryl, O-acyl, O-heterocylic,O-heteroraryl, O-alkenylcarbonyl and derivatives thereof; R2 is selectedfrom hydrogen, hydroxyl, O-angeloyl, O-tigloyl, O-senecioyl, O-alkyl,O-dibenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl, O-benzoyl alkylsubstituted O-alkanoyl, O-alkanoyl substituted phenyl, O-alkenoylsubstituted phenyl, O-aryl, O-acyl, O-heterocylic, O-heteroraryl,O-alkenylcarbonyl and derivatives thereof; R4 represents CH3, CHO, CH₂R6or COR6, wherein R6 is selected from hydroxyl, O-angeloyl, O-tigloyl,O-senecioyl, O-alkyl, O-dibenzoyl, O-benzoyl, O-alkanoyl, O-alkenoyl,O-benzoyl alkyl substituted O-alkanoyl, O-alkanoyl substituted phenyl,O-alkenoyl substituted phenyl, O-aryl, O-acyl, O-heterocylic,O-heteroraryl, O-alkenylcarbonyl and derivatives thereof; R3 is H or OH;R5 is H or OH, particularly OH; wherein R6, R7, R8, R9, R10, R11, R12are independently attached a group selecting from CH₃, CH₂OH, CHO, COOH,COO-alkyl, COO-aryl, COO-heterocyclic, COO-heteroaryl, CH₂Oaryl,CH₂O-heterocyclic, CH₂O-heteroaryl, alkyls group, hydroxyl, acetylgroup; R13 is COOH or COO-alkyl; In an embodiment, the compound is inform of salts.

also named (1L),wherein R1, R2 are angeloyl, R3 is OH, R4 is CH2OH, R5 is H or OH. In anembodiment the method comprises administering to a subject or contactingthe cells with the compounds of following:

or3-O-[β-D-galactopyranosyl(1→2)]-β-D-xyopyranosyl(1→3)-β-D-glucuronopyranosyl-21-O-angeloyl, 22-O-angeloyl-3β, 15α, 16α,21β, 22α, 28-hexahydroxyolean-12-ene,

or3-O-[β-D-galactopyranosyl(1→2)]-β-D-xyopyranosyl(1→3)-β-D-glucuronopyranosyl-21-O-benzoyl, 22-O-benzoyl-3β, 15α, 16α,21β, 22α, 28-hexahydroxyolean-12-ene

or3-O-[β-D-galactopyranosyl(1→2)]-β-D-xyopyranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(2-methylpropanoyl)-O-angeloyl,22-O-(2-methylbutanoyl)-3β, 15α, 16α, 21β, 22α,28-hexahydroxyolean-12-ene.

A sugar moiety is a segment of a molecule comprising one or more sugargroups. Substitution, deletion and/or addition of any group in theabove-described compounds will be apparent to one of ordinary skill inthe art based on the teaching of this application. In a furtherembodiment, the substitution, deletion and/or addition of the group(s)in the compound of the invention does not substantially affect thebiological function of the compound.

This invention provides a method or a use of compound for manufacture ofmedicament for inhibiting venous insufficiency, particularly hemorrhoidsor inhibiting leg swelling, or peripheral edema; antilipemic; fortreating chronic venous disease, varicose vein disease, varicosesyndrome, venous stasis, expectorant, peripheral vascular disorders,cerebro-organic convulsion, cerebral circulation disorder, cerebraledema, psychoses, dysmenorrheal, hemorrhoids, episiotomies, hamonhoids,peripheral edema formation or postoperative swelling; for reducingsymptoms of pain; for reducing symptoms of stomach pain; for reducingsymptoms of leg pain; for treating pruritis, lower leg volume,thrombosis, thromophlebitis; for preventing gastric ulcers antispasmoticcomprising administering to a subject, in need thereof, an effectiveamount of the composition of any one of the above compounds or acompound comprises a triterpene which comprises any two of angeloyl,tigloyl, senecioyl, preferably two angeloyl groups, and a sugar moiety,glucose, galactose, rhamnose, arabinose, xylose, fucose, allose,altrose, gulose, idose, lyxose, mannose, psicose, ribose, sorbose,tagatose, talose, fructose, alduronic acid, glucuronic acid orgalacturonic acid, or a derivative thereof, or combinations thereof,preferably selected from glucuronic acid, galacturonic acid, glucose,galactose and arabinose. The method regulates or interacts with adhesionprotein, wherein the adhesion proteins comprise fibronectin, integrinsfamily, myosin, vitronectin, collagen, laminin, glycosylation cellsurface proteins, polyglycans, cadherin, heparin, tenascin, CD 54, CAM,elastin and FAK. In an embodiment, the method comprises regulating thesecretion of fibronectin. In an embodiment, the method comprisesinhibiting leishmaniases, modulating adhesion or angiogenesis of cancercells, antiparasitics or manufacturing an adjuvant composition. In anembodiment, antiparasitics comprise inhibiting leishmaniases,amoebiasis, trypanosomiasis, toxoplasmosis or malaria.

This invention provides a method for inhibiting the growth, migration,metastasis of cancer by altering the characteristics of membranes ofcancer cell, wherein the method comprises reducing adhesion protein;wherein the adhesion proteins comprise fibronectin, integrins family,myosin, vitronectin, collagen, laminin, glycosylation cell surfaceproteins, polyglycans, cadherin, heparin, tenascin, CD 54, CAM, elastinand FAK; wherein the method comprises inhibiting the secretion offibronectin, wherein the methods comprises administering to a subject,in need thereof, an appropriate amount of triterpenoidal saponinscomprising two or more angeloyl groups, or a compound comprising atriterpene which comprises of any two of angeloyl, tigloyl, senecioyl,preferably two angeloyl groups, and a sugar moiety, glucose, galactose,rhamnose, arabinose, xylose, fucose, allose, altrose, gulose, idose,lyxose, mannose, psicose, ribose, sorbose, tagatose, talose, fructose,alduronic acid, glucuronic acid or galacturonic acid, or a derivativethereof, or combinations thereof, preferably selected from glucuronicacid, galacturonic acid, glucose, galactose and arabinose. Thisinvention provides a composition comprising an effective amount of thecompound of any one of compound selected from the above formula or asalt, ester, metabolite or derivative thereof as a medicament forreducing expression and secretion of adhesion proteins; wherein theadhesion proteins comprise fibronectin, integrins family, myosin,vitronectin, collagen, laminin, glycosylation cell surface proteins,polyglycans, cadherin, heparin, tenascin, CD 54, CAM, elastin and FAK,for inhibiting the growth, migration, metastasis of cancer, wherein thecancers comprise breast cancer, leukocytic cancer, liver cancer, ovariancancer, bladder cancer, prostatic cancer, skin cancer, bone cancer,brain cancer, leukemia cancer, lung cancer, colon cancer, CNS cancer,melanoma cancer, renal cancer, cervical cancer, esophageal cancer,testicular cancer, spleenic cancer, kidney cancer, lymphatic cancer,pancreatic cancer, stomach cancer and thyroid cancer.

This invention also provides a composition comprising the abovedescribed compounds or their derivatives for reducing adhesion protein,wherein the adhesion proteins comprise fibronectin, integrins family,myosin, vitronectin, collagen, laminin, glycosylation cell surfaceproteins, polyglycans, cadherin, heparin, tenascin, CD 54, CAM, elastinand FAK; wherein comprising inhibiting the secretion of fibronectin,wherein the composition is used for treating venous insufficiency,particularly hemorrhoids or inhibiting leg swelling, or peripheraledema, lipemic, chronic venous disease, varicose vein disease, varicosesyndrome, venous stasis, Expectorant, peripheral vascular disorders,cerebro-organic convulsion, cerebral circulation disorder, cerebraledema, psychoses, dysmenorrheal, episiotomies, hemonhoids, peripheraledema formation or postoperative swelling; for reducing symptoms ofpain; for reducing symptoms of stomach pain; for reducing symptoms ofleg pain; for treating pruritis, lower leg volume, thrombosis,thromophlebitis; for preventing gastric ulcers antispasmotic, inhibitingleishmaniases; for modulating adhesion or angiogenesis of cancer cells;antiparasitics or manufacturing an adjuvant composition. In anembodiment of the above, the uses of compositions comprising any one oftriterpenoid saponins with the following formula:

3-O-{[β-D-galactopyranosyl(1→2)]-[α-L-arabinofuranosyl(1→3)]-β-D-glucuronopyranoside butylester}-21-O-acetyl-22-O-angeloyl-3β,16α,21β,22α,28-pentahydroxyolean-12-ene,

3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β, 15α, 16α, 21β, 22α,28-hexahydroxyolean-12-ene,

3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β,16α, 21β, 22α, 28-pentahydroxyolean-12-ene,

3-O-[β-D-glucopyranosyl-(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α, 16α, 21β, 22α, 24β, 28-heptahydroxyolean-12-ene,

3-O-[β-glucopyranosyl (1→2)]-α-arabinofuranosyl(1→3)-β-glucuronopyranosyl-21, 22-O-diangeloyl-3β, 16α, 21β, 22α, 24β,28-hexahydroxyolean-12-ene,

3-O-[β-galactopyranosyl(1→2)]-α-arabinofuranosyl(1→3)-β-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-rhamnopyranosyl-28-O-acetyl-3β,16α, 21β, 22α, 28-pentahydroxyolean-12-ene,

3-O-[β-galactopyranosyl(1→2)]-α-arabinofuranosyl(1→3)-β-glucuronopyranosyl-21, 22-O-diangeloyl-3β, 16α, 21β, 22α,28-pentahydroxyolean-12-ene,

3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-angeloyl,22-O-(2-methylpropanoyl)-3β, 15α, 16α, 21β, 22α,28-hexahydroxyolean-12-ene,

3-O-[β-D-galactopyranosyl-(1→2)]-α-L-arabinofuranosyl-(1→3)-β-D-glucuronopyranosyl-21-O-angeloyl-28-O-2-methylbutanoyl-3β,15α, 16α, 21β, 22α, 28-hexahydroxyolean-12-ene

This invention also provides a composition for regulating or reducingadhesion proteins, wherein the adhesion proteins comprise fibronectin,integrins family, myosin, vitronectin, collagen, laminin, glycosylationcell surface proteins, polyglycans, cadherin, heparin, tenascin, CD 54,CAM, elastin and FAK; inhibiting venous insufficiency, particularlyhemorrhoids or inhibition of leg swelling, or inhibiting cancer growth,inhibiting leishmaniases, modulating adhesion of cancer cells,modulating angiogenesis of cancer cells, antiparasitics or manufacturingan adjuvant composition, comprising any of compounds selected from thefollowing compounds (A) to (X) and (A1) to (X1) incorporated here fromPCT/US2008/002086, 1188-ALA-PCT:

In an embodiment, a triterpene comprising the following structure hasactivities of reducing adhesion proteins to block the migration,inhibiting metastasis of cancer cells, inhibiting growth of cancers,inhibiting leishmaniases, modulating adhesion or angiogenesis of cancercells, antiparasitics, or manufacturing an adjuvant composition.

wherein at least two of R1, R2 and R3 comprise compounds selected fromangeloyl, acetyl, tigloyl, senecioyl, benzoyl, dibenzoyl, alkanoyl,alkenoyl, benzoyl alkyl substituted alkanoyl, aryl, heterocylic,heteroraryl, alkanoyl substituted phenyl, alkenoyl substituted phenyl,alkenylcarbonyl or substituted with an C2-9 acid or derivative thereof.In an embodiment, at least one of R1, R2 and R3 comprise a sugar moietycomprising two compounds selected from angeloyl, acetyl, tigloyl,senecioyl, alkyl, benzoyl, dibenzoyl, alkanoyl, alkenoyl, benzoyl alkylsubstituted alkanoyl, aryl, acyl, heterocylic, heteroraryl, alkanoylsubstituted phenyl, alkenoyl substituted phenyl, alkenylcarbonyl orsubstituted with an C2-9 acid or derivative thereof. In embodiment, R1,R2 or R3 comprise angeloyl groups, tigloyl groups, senecioyl groups oracetyl group or their combinations, preferable wherein at least two ofthe R1, R2 and R3 comprise angeloyl groups. In an embodiment, R5comprises sugar moiety. In an embodiment, the sugar moiety comprises atleast one sugar, or glucose, or galactose, or rhamnose, or arabinose, orxylose, or alduronic acid, or glucuronic acid, or galacturonic acid, ortheir derivative thereof, or the combination thereof. In an embodiment,the sugar moiety comprises one or more sugar selected from, but is notlimited to glucose, galactose, rhamnose, arabinose, xylose, fucose,allose, altrose, gulose, idose, lyxose, mannose, psicose, ribose,sorbose, tagatose, talose, fructose, alduronic acid, glucuronic acid,galacturonic acid, or derivatives thereof, or the combination thereof.In an embodiment, the sugar moiety comprises glucose, galactose orarabinose, or combination thereof, or derivatives thereof. In anembodiment, the sugar moiety comprise alduronic acids, galactose andarabinose, wherein the alduronic comprise glucuronic acid orgalacturonic acid. In an embodiment, the sugar moiety comprisesalduronic acids, glucose and arabinose, wherein the alduronic compriseglucuronic acid or galacturonic acid. In an embodiment, R5 is Hydrogen.In an embodiment, the R1, R 2 and R3 may be attached in other positionof the structure. In an embodiment, the compound is a triterpenoidsaponin comprising at least two angeloyl groups, tigloyl groups,senecioyl groups or acetyl group or their combinations, preferably withat least two angeloyl groups. In an embodiment, at least two groups areselected from angeloyl, acetyl, tigloyl, senecioyl, benzoyl, dibenzoyl,alkanoyl, alkenoyl, benzoyl alkyl substituted alkanoyl, aryl, acyl,heterocylic, heteroraryl, alkanoyl substituted phenyl, alkenoylsubstituted phenyl, alkenylcarbonyl or substituted with an C2-9 acid orderivative thereof. In an embodiment, at least one of the side bonds ofthe compound comprises a sugar moiety comprising two compounds selectedfrom angeloyl, acetyl, tigloyl, senecioyl, benzoyl, dibenzoyl, alkanoyl,alkenoyl, benzoyl alkyl substituted alkanoyl, aryl, acyl, heterocylic,heteroraryl, alkanoyl substituted phenyl, alkenoyl substituted phenyl,alkenylcarbonyl or substituted with an C2-9 acid or derivative thereof.In an embodiment, the compound comprises a sugar moiety. In a furtherembodiment, the sugar moiety comprises glucose, galactose or arabinoseor combination thereof. In a further embodiment, the sugar moietycomprises at least one sugar, or glucose, or galactose, or rhamnose, orarabinose, or xylose, or alduronic acid, or glucuronic acid, orgalacturonic acid, or their derivative thereof, or the combinationsthereof. In a further embodiment, the sugar moiety comprises one or moresugars selected from, but is not limited to glucose, galactose,rhamnose, arabinose, xylose, fucose, allose, altrose, gulose, idose,lyxose, mannose, psicose, ribose, sorbose, tagatose, talose, fructose,alduronic acid, glucuronic acid, galacturonic acid, or derivativesthereof, or combinations thereof.

A composition comprises an effective amount of compound selected fromthe above formula or a salt, ester, metabolite or derivative thereof asa medicament for regulating or reducing adhesion protein, blocking themigration, metastasis of cancer cells, inhibiting tumor or cancer cellgrowth and for treating cancer, wherein the cancers comprise breastcancer, leukocytic cancer, liver cancer, ovarian cancer, bladder cancer,prostatic cancer, skin cancer, bone cancer, brain cancer, leukemiacancer, lung cancer, colon cancer, CNS cancer, melanoma cancer, renalcancer, cervical cancer, esophageal cancer, testicular cancer, spleeniccancer, kidney cancer, lymphatic cancer, pancreatic cancer, stomachcancer and thyroid cancer.

In a further embodiment, a compound or sapongenin comprising thestructure (d) or (e) has anti-cancer or virus inhibiting activities.

A composition for regulating or reducing adhesion proteins, blocking themigration or metastasis of cancer cells, treating cancers or inhibitingviruses, comprises a compound, wherein the compound is a triterpene,which comprises at least two side chains which comprise angeloyl groups,wherein the side chains are at adjacent carbon in trans position. In anembodiment, the side chains are at alternate carbon in cis position. Inan embodiment, the side chains are at alternate carbon in transposition. In an embodiment, the side chains are attached to an acyl. Inan embodiment, the side chains are attached to an unsaturated group. Inan embodiment, the side chains are in non-adjacent carbon cis or transposition. In an embodiment, the side chains comprise a functional groupcapable of performing the function of angeloyl group.

The above compounds can be used for regulating or reducing adhesionproteins, blocking the migration or metastasis of cancer cells,inhibiting tumor cell growth, or reducing leg swelling, symptoms ofchronic venous insufficiency, peripheral edema, antilipemic, chronicvenous disease, varicose vein disease, varicose syndrome, venous stasis,expectorant, peripheral vascular disorders, by administering to asubject in need thereof, an effective amount of the above describedcompounds.

This invention provides a method for inhibiting tumor cell growth,regulating cell growth, reducing inflammation, inhibiting leishmaniases,modulating adhesion or angiogenesis of cancer cells, antiparasitics ormanufacturing an adjuvant composition, comprising administering to asubject, in need thereof, an effective amount of the compound whichcomprises any of the above structures to said subject. The cancers areincluded but not limited to breast cancer, leukocytic cancer, livercancer, ovarian cancer, bladder cancer, prostatic cancer, skin cancer,bone cancer, brain cancer, leukemia cancer, lung cancer, colon cancer,CNS cancer, melanoma cancer, renal cancer, cervical cancer, esophagealcancer, testicular cancer, spleenic cancer, kidney cancer, lymphaticcancer, pancreatic cancer, stomach cancer and thyroid cancer. Thisinvention also provides a method for reducing swelling, reducingsymptoms of chronic venous insufficiency, peripheral edema, chronicvenous disease, varicose vein disease, varicose syndrome, venous stasis,Expectorant, peripheral vascular disorders, cerebro-organic convulsion,cerebral circulation disorder, cerebral edema, psychoses, dysmenorrheal,hemorrhoids, episiotomies, peripheral edema formation or postoperativeswelling; for reducing symptoms of leg pain; for treating pruritis,lower leg volume, for reducing symptoms of pain; thrombosis,thromophlebitis; for preventing gastric ulcers antispasmotic,antilipemic, comprising administering to a subject, in need thereof, aneffective amount of the composition of this invention.

This invention provides a composition comprising the compounds providedin the invention for treating cancers; for inhibiting virus; forpreventing cerebral aging; for improving memory; improving cerebralfunctions, for curing enuresis, frequent micturition, urinaryincontinence, dementia, Alzheimer's disease, autism, brain trauma,Parkinson's disease or other diseases caused by cerebral dysfunctions;for treating arthritis, rheumatism, poor circulation, arteriosclerosis,Raynaud's syndrome, angina pectoris, cardiac disorder, coronary heartdisease, headache, dizziness, kidney disorder; cerebrovascular disease;inhibiting NF-Kappa B activation; for treating brain edema, sever acuterespiratory syndrome, respiratory viral diseases, chronic venousinsufficiency, hypertension, chronic venous disease, hemonhoids,peripheral edema formation, varicose vein disease, flu, post traumaticedema and postoperative swelling; for inhibiting blood clot, forinhibiting ethanol absorption; for lowering blood sugar; for regulatingthe adrenocorticotropin and corticosterone level. This inventionprovides a composition for AntiMS, antianeurysm, antiasthmatic,antibradykinic, anticapillarihemorrhagic, anticephalagic,anticervicobrachialgic, antieclamptic, antiedemic, antiencaphalitic,antiepiglottitic, antiexudative, antiflu, antifracture, antigingivitic,antihematomic, antiherpetic, antihistaminic, antihydrathritic,antimeningitic, antioxidant, antiperiodontic, antiphlebitic,antipleuritic, antiraucedo, antirhinitic, antitonsilitic, antiulcer,antivaricose, antivertiginous, anti-oedematous, anti inflammatory,cancerostatic, corticosterogenic, diuretic, fungicide, hemolytic,hyaluronidase inhibitor, lymphagogue, natriuretic, pesticide, pituitarystimulant, thymolytic, vasoprotective, inhibiting leishmaniases,modulating adhesion or angiogenesis of cancer cells, antiparasitics, ormanufacturing an adjuvant composition and venotonic treatment.

This invention provides a use of compounds or methods for inhibiting theexpression or secretion of adhesion proteins of cancers, cancer cellmigration, metastasis or growth of cancers, wherein this inventioncomprises a process and method for administration of the an effectiveamount of composition, wherein administration is by intravenousinjection, intravenous drip, intraperitoneal injection or oraladministration; wherein administration is by intravenous drip:0.003-0.03 mg/kg body weight of compound dissolved in 250 ml of 10%glucose solution or in 250 ml of 0.9% NaCl solution, or by intravenousinjection: 0.003-0.03 mg/kg body weight per day of compound dissolved in10-20 ml of 10% glucose solution or of 0.9% NaCl solution, or 0.01-0.03mg/kg body weight of compound dissolved in 250 ml of 10% glucosesolution or in 250 ml of 0.9% NaCl solution, or by intravenousinjection: 0.01-0.03 mg/kg body weight per day of compound dissolved in10-20 ml of 10% glucose solution or of 0.9% NaCl solution, or 0.01-0.05mg/kg body weight of compound dissolved in 250 ml of 10% glucosesolution or in 250 ml of 0.9% NaCl solution, or by intravenousinjection: 0.01-0.05 mg/kg body weight per day of compound dissolved in10-20 ml of 10% glucose solution or of 0.9% NaCl solution, or 0.05-0.2mg/kg body weight of compound dissolved in 250 ml of 10% glucosesolution or in 250 ml of 0.9% NaCl solution, or by intravenousinjection: 0.05-0.2 mg/kg body weight per day of compound dissolved in10-20 ml of 10% glucose solution or of 0.9% NaCl solution, or byintravenous drip: 0.1-0.2 mg/kg body weight per day of compounddissolved in 250 ml of 10% glucose solution or in 250 ml of 0.9% NaClsolution, or by intravenous injection: 0.1-0.2 mg/kg body weight per daycompound dissolved in 10-20 ml of 10% glucose solution or of 0.9% NaClsolution, or by intraperitoneal injection (I.P.): 2.5 mg/kg body weightper day compound dissolved in 10% glucose solution or of 0.9% NaClsolution, or by oral administration wherein the dosage of mammal is0.01-3 mg/kg, 0.1-5 mg/kg, 1-10 mg/kg, 10-30 mg/kg, 30-60 mg/kg, or60-90 mg/kg body weight of compound, or by intravenous injection orintravenous drip wherein the dosage of mammal is 0.01-0.1 mg/kg, 0.1-0.2mg/kg, 0.2-0.4 mg/kg, or 0.4-0.6 mg/kg body weight of compound, or byintraperitoneal injection (I.P.) wherein the dosage of mammal is 1-3mg/kg, 3-5 mg/kg, 4-6 mg/kg, or 6-10 mg/kg body weight of compound.

This invention provides a use of compounds or methods for inhibiting theexpression or secretion of adhesion proteins of cancers, cancer cellmigration, metastasis or growth of cancers, wherein the inventioncomprises a pharmaceutical composition comprising the compound of thisinvention or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier or diluent, wherein said compound ispresent in a concentration of 0.01 ug/ml to 65 ug/ml, or wherein saidcompound is present in a concentration of 0.01 ug/ml to 40 ug/ml, orwherein said compound is present in a concentration of 0.01 ug/ml to 30ug/ml, or wherein said compound is present in a concentration of 0.011ug/ml to 10 ug/ml, or wherein said compound is present in aconcentration of 0.011 ug/ml to 5 ug/ml, or wherein said compound ispresent in a concentration of 5 ug/ml to 10 ug/ml, or wherein saidcompound is present in a concentration of 0.1 ug/ml to 5 ug/ml, orwherein said compound is present in a concentration of 0.1 ug/ml to 7.5ug/ml, or wherein said compound is present in a concentration of 0.1ug/ml to 10 ug/ml, or wherein said compound is present in aconcentration of 0.1 ug/ml to 15 ug/ml, or wherein said compound ispresent in a concentration of 0.1 ug/ml to 20 ug/ml, or wherein saidcompound is present in a concentration of 0.1 ug/ml to 30 ug/ml, orwherein said compound is present in a concentration of 1 ug/ml to 5ug/ml, or wherein said compound is present in a concentration of 1 ug/mlto 7.5 ug/ml, or wherein said compound is present in a concentration of1 ug/ml to 10 ug/ml, or wherein said compound is present in aconcentration of 1 ug/ml to 15 ug/ml, or wherein said compound ispresent in a concentration of 1 ug/ml to 20 ug/ml, or wherein saidcompound is present in a concentration of 1 ug/ml to 30 ug/ml, orwherein said compound is present in a concentration of 3 ug/ml to 5ug/ml, or wherein said compound is present in a concentration of 3 ug/mlto 7.5 ug/ml, or wherein said compound is present in a concentration of3 ug/ml to 10 ug/ml, or wherein said compound is present in aconcentration of 3 ug/ml to 15 ug/ml, or wherein said compound ispresent in a concentration of 3 ug/ml to 20 ug/ml, or wherein saidcompound is present in a concentration of 3 ug/ml to 30 ug/ml, orwherein said compound is present in a concentration of 4 ug/ml to 5ug/ml, or wherein said compound is present in a concentration of 4 ug/mlto 7.5 ug/ml, or wherein said compound is present in a concentration of4 ug/ml to 10 ug/ml, or wherein said compound is present in aconcentration of 4 ug/ml to 15 ug/ml, or wherein said compound ispresent in a concentration of 4 ug/ml to 20 ug/ml, or wherein saidcompound is present in a concentration of 4 ug/ml to 30 ug/ml, orwherein said compound is present in a concentration of 5 ug/ml to 8ug/ml, or wherein said compound is present in a concentration of 5 ug/mlto 9 ug/ml, or wherein said compound is present in a concentration of 5ug/ml to 10 ug/ml, or wherein said compound is present in aconcentration of 5 ug/ml to 15 ug/ml, or wherein said compound ispresent in a concentration of 5 ug/ml to 20 ug/ml, or wherein saidcompound is present in a concentration of 5 ug/ml to 30 ug/ml, orwherein said compound is present in a concentration of 7 ug/ml to 8ug/ml, or wherein said compound is present in a concentration of 7 ug/mlto 9 ug/ml, or wherein said compound is present in a concentration of 7ug/ml to 10 ug/ml, or wherein said compound is present in aconcentration of 7 ug/ml to 15 ug/ml, or wherein said compound ispresent in a concentration of 7 ug/ml to 20 ug/ml, or wherein saidcompound is present in a concentration of 7 ug/ml to 30 ug/ml.

This invention provides a use of compounds or methods for inhibiting theexpression or secretion of adhesion proteins of cancers, cancer cellmigration, metastasis or growth of cancers, wherein the inventioncomprises a pharmaceutical composition comprising the compound of thisinvention or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier or diluent, wherein said compound ispresent in a concentration of 0.008 uM to 80 uM, or wherein saidcompound is present in a concentration of 0.01 uM to 60 uM, or whereinsaid compound is present in a concentration of 0.01 uM to 50 uM, orwherein said compound is present in a concentration of 0.01 uM to 40 uM,or wherein said compound is present in a concentration of 0.01 uM to 30uM, or wherein said compound is present in a concentration of 0.01 uM to20 uM, or wherein said compound is present in a concentration of 0.01 uMto 10 uM, or wherein said compound is present in a concentration of 5 uMto 10 uM, or wherein said compound is present in a concentration of 0.1uM to 5 uM, or wherein said compound is present in a concentration of0.1 uM to 7.5 uM, or wherein said compound is present in a concentrationof 0.1 uM to 10 uM, or wherein said compound is present in aconcentration of 0.1 uM to 15 uM, or wherein said compound is present ina concentration of 0.1 uM to 20 uM, or wherein said compound is presentin a concentration of 0.1 uM to 30 uM or wherein said compound ispresent in a concentration of 0.1 uM to 40 uM, or wherein said compoundis present in a concentration of 0.1 uM to 50 uM or wherein saidcompound is present in a concentration of 0.1 uM to 60 uM, or whereinsaid compound is present in a concentration of 0.1 uM to 80 uM, orwherein said compound is present in a concentration of 1 uM to 5 uM, orwherein said compound is present in a concentration of 1 uM to 7.5 uM,or wherein said compound is present in a concentration of 1 uM to 10 uM,or wherein said compound is present in a concentration of 1 uM to 15 uM,or wherein said compound is present in a concentration of 1 uM to 20 uM,or wherein said compound is present in a concentration of 1 uM to 30 uMor wherein said compound is present in a concentration of 1 uM to 40 uM,or wherein said compound is present in a concentration of 1 uM to 50 uMor wherein said compound is present in a concentration of 1 uM to 60 uM,or wherein said compound is present in a concentration of 1 uM to 80 uM,or wherein said compound is present in a concentration of 3 uM to 5 uM,or wherein said compound is present in a concentration of 3 uM to 7.5uM, or wherein said compound is present in a concentration of 3 uM to 10uM, or wherein said compound is present in a concentration of 3 uM to 15uM, or wherein said compound is present in a concentration of 3 uM to 20uM, or wherein said compound is present in a concentration of 3 uM to 30uM or wherein said compound is present in a concentration of 3 uM to 40uM, or wherein said compound is present in a concentration of 3 uM to 50uM or wherein said compound is present in a concentration of 3 uM to 60uM, or wherein said compound is present in a concentration of 3 uM to 80uM, or wherein said compound is present in a concentration of 5 uM to 8uM, or wherein said compound is present in a concentration of 5 uM to 10uM, or wherein said compound is present in a concentration of 5 uM to 15uM, or wherein said compound is present in a concentration of 5 uM to 20uM, or wherein said compound is present in a concentration of 5 uM to 30uM or wherein said compound is present in a concentration of 5 uM to 40uM, or wherein said compound is present in a concentration of 5 uM to 50uM or wherein said compound is present in a concentration of 5 uM to 60uM, or wherein said compound is present in a concentration of 5 uM to 80uM. or wherein said compound is present in a concentration of 7 uM to 8uM, or wherein said compound is present in a concentration of 7 uM to 10uM, or wherein said compound is present in a concentration of 7 uM to 15uM, or wherein said compound is present in a concentration of 7 uM to 20uM, or wherein said compound is present in a concentration of 7 uM to 30uM or wherein said compound is present in a concentration of 7 uM to 40uM, or wherein said compound is present in a concentration of 7 uM to 50uM or wherein said compound is present in a concentration of 7 uM to 60uM, or wherein said compound is present in a concentration of 7 uM to 80uM.

Alkenyl means unsaturated linear or branched structures and combinationsthereof, having 1-7 carbon atoms, one or more double bonds therein.Non-limiting examples of alkenyl groups include vinyl, propenyl,isopropenyl, butenyl, s- and t-butenyl, pentenyl, hexenyl, butadienyl,pentadienyl, and hexadienyl.

An aryl is a functional group of organic molecules derived from anaromatic compound such as benzene, a 6-14 membered carbocyclic aromaticring system comprising 1-3 benzene rings. If two or more aromatic ringsare present, then the rings are fused together, so that adjacent ringsshare a common bond. Examples include phenyl and naphthyl. The arylgroup may be substituted with one or more substitutes independentlyselected from halogen, alkyl or alkoxy. Acyl is a functional groupobtained from an organic acid by the removal of the carboxyl. Acylgroups can be written as having the general formula —COR, where there isa double bond between the carbon and oxygen. The names of acyl groupstypically end in -yl, such as formyl, acetyl, propionyl, butyryl andbenzoyl.

Benzoyl is one of acyls, C₆H₅COR, obtained from benzoic acid by theremoval of the carboxyl.

Heterocyclic compound—a compound containing a heterocyclic ring whichrefers to a non-aromatic ring having 1-4 heteroatoms said ring beingisolated or fused to a second ring selected from 3- to 7-memberedalicyclic ring containing 0-4 heteroatoms, aryl and heteroaryl, whereinsaid heterocyclic comprises pyrrolidinyl, pipyrazinyl, morpholinyl,trahydrofuranyl, imidazolinyl, thiomorpholinyl.

Heterocyclyl groups derived from heteroarenes by removal of a hydrogenatom from any ring atom.

Alkanoyl is the general name for an organic functional group RCO—, whereR represents hydrogen or an alkyl group. Preferably alkanoyl is selectedfrom acetyl, propionoyl, butyryl, isobutyryl, pentanoyl and hexanoyl.

Alkenoyl is alkenylcarbonyl in which alkenyl is defined above. Examplesare pentenoyl(tigloyl) and hexenoyl(angeloyl).

Alkyl is a radical containing only carbon and hydrogen atoms arranged ina chain, branched, cyclic or bicyclic structure or their combinations,having 1-18 carbon atoms. Examples include but are not limited tomethyl, ethyl, propyl isopropyl, butyl, s- and t-butyl, pentyl, hexyl,heptyl, octyl, nonyl, undecyl, dodecyl, tridecyl, tetradecyl,pentadecyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

Benzoyl alkyl substituted alkanoyl is refer to straight or branchedC1-C6 alkanoyl substituted with at least one benzoyl and at least onealkyl, wherein the benzoyl is attached to a straight or branched C1-C6alkyl. Preferably a benzoyl alkyl substituted alkanoyl is benzoyl methylisobutanoyl.

A sugar moiety is a segment of molecule comprising one or more sugars orderivatives thereof or alduronic acid thereof.

Isobutyryl is Synonym of 2-Methylpropanoyl; (Y)Y3, Y and Y3 representthe same compound; YM and (ACH-Y) represent the same compound.

Salts include sodium salts.

This invention provides a method of altering the characteristic ofcancer cell membrane to block the migration, metastasis of cancer cellsor inhibit the growth of cancers or anti-angiogenesis.

This invention provides a composition and method for inhibiting thegrowth, migration, metastasis of cancer by altering the adhesioncharacteristic of membrane of cancer cell, wherein the cancers comprisebreast cancer, leukocyte cancer, liver cancer, ovarian cancer, bladdercancer, prostate cancer, skin cancer, bone cancer, brain cancer,leukemia cancer, lung cancer, colon cancer, CNS cancer, melanoma cancer,renal cancer or cervix cancer, wherein the method comprisesadministering to a subject or contacting the cells with Xanifolia Y0,Y1, Y2, Y, Y5, Y7, Y8, Y9, Y10, or a salt, ester, metabolite thereof. Inan embodiment the method comprises administering to a subject orcontacting the cells with the compound selected from formula in thisapplication.

This application shows Xanifolia-Y is an alternate or supplemental agentto DNA-inhibition or microtubule-targeting drugs. It could be beneficialif it is used singly or in combination with other drugs of differentmechanisms (block M-phase progression or DNA synthesis). Our inventionsshow combined effect of Xanifolia-Y and paclitaxel on inhibition of ES2cells' growth (Details in Experiment 15).

Identify the binding target of Xanifolia-Y of adhesion proteins andsignaling proteins in ovarian cancer cells.

In our animal studies, it was shown that Xanifolia-Y extended the lifespan of tumor bearing mice. (Reference Experiments 7, 8, 9 in U.S. Ser.No. 11/683,198, filed on Mar. 7, 2007,). The animals died sooner if thetreatment of Xanifolia-Y was delayed (comparing results of treatmentsstarted from 1, 4 or 10 days after tumor inoculation). The results showthat Xanifolia-Y inhibits migration or metastasis of the inoculatedcancer cells. Ovarian carcinoma cells express high levels of adhesionmolecules. Adhesion proteins are present in both cancer cells andmesothelial cells. While the loss of adhesion blocks of the proteinaccessibility due to a result of modulating by Xanifolia-Y. In anembodiment, the interaction of Xanifolia-Y with membrane alter theadhesion protein's binding site(s).

Fibronectin is a kind of glycoprotein that binds to membrane spanningreceptor proteins comprising the integrins, collagen, fibrin and heparinsulfate. Fibronectin has been implicated in tumor development andmetastasis. This application provides methods and compositions formodulating the gene expression of fibronectin, inhibiting the secretionof fibronectin, reducing the receptors of fibronectin, reducing theadhesion ability fibronectin, inhibiting the metastasis, or inhibitingcancer growth, wherein the method and composition comprisesadministering to the said subject an effective amount of compoundsselected in this application.

Angiogenesis is a process involving the growth of new blood vessels. Itis a normal process in growth and development. The angiopoietins areprotein growth factors that modulate angiogenesis. The identifiedangiopoietins comprise angiopoietin 1, angiopoietin 2, angiopoietin 3,angiopoietin 4, angiopoietin 5, angiopoietin 6, angiopoietin 7,angiopoietin-like 1, angiopoietin-like 2, angiopoietin-like 3,angiopoietin-like 4, angiopoietin-like 5, angiopoietin-like 6, andangiopoietin-like 7. In an embodiment, the angiopoietin 1 is a positivefactor to promote the new blood vessels. In embodiment, the angiopoietin2 is antagonist of angiopoietin 1, which is a negative factor for thegrowth of new blood vessels. This application provides methods andcompositions for modulating angiopoietin and inhibiting cancer growth;wherein the cancers comprise breast, leukocyte, liver, ovarian, bladder,prostate, skin, bone, brain, leukemia, lung, colon, CNS, melanoma, renaland cervix cancer, wherein the methods and compositions compriseadministering to the said subject as effective amount of compoundsselected in this application. The compounds in this application arepositive regulating angiopoietin 2. The compounds in this applicationare negative regulating the angiopoietin 1. The results of the microarray experiment showed that compound Y and YM (ACH-Y) modulate the geneexpression of angiopoietin family in ES2 cells. They promoteangiopoietin 2 and inhibit angiopoietin 1 and angiopoietin-like 1 andangiopoietin-like 4.

The compounds in this application are used antiparasitics, enhancing animmune response, providing adjuvant activities or providing vaccineactivities, anti-angiogenesis, inhibiting cancer cell metastasis andinhibiting cancer growth, wherein the compounds comprise Xanifolia Y0,Y1, Y2, Y, Y7, Y8, Y9, Y10, ACH-Y or a salt, ester, metabolite thereofand compounds selected from formula (1A), (1B), (1C), (1D), (1E), (1F),(1G), (1H), (1J), (1K), (1L). In an embodiment the method isadministering contacting the compound in this application comprisingXanifolia Y0, Y1, Y2, Y, Y7, Y8, Y9, Y10, Xanifolia (x), Escin or Aescinor a salt, ester, metabolite thereof. In an embodiment the compound maybe selected from formulas (1A), (1B), (1C), (1D), (1E), (1F), (1G),(1H), (1J), (1K), (1L). In an embodiment, the compound comprises atriterpene backbone, two angeloyl groups and sugar moiety. In anembodiment the compound(s) are selected from Compound A to X and A1 toX1 in the application. In an embodiment the compound(s) are selectedfrom Compound Z1 to Z7 in the application. In an embodiment thecompound(s) are selected from ACH-Z4, ACH-Y10, ACH-Y2, ACH-Y8, ACH-Y7,ACH-Y0, ACH-X, ACH-E. In an embodiment the saponins comprise Ba1, Ba2,Ba3, Ba4, Ba5, Ba6, Ba7, Ba8, Ba9, Ba10, Ba11, Ba12, Ba13, Ba14, Ba15,Ba16, Ba 17. The triterpene compounds of this invention can beadministered to a subject in need thereof treating the subject, whereinincluding preventing cancer or has adjuvant effect to the subject, orinhibiting the initiation or promotion of cancer, or killing thecancer/tumor cells. In an embodiment the compounds inhibit theactivation of nuclear factor-kB, wherein inhibiting the localization orwherein binding the DNA. In an embodiment the compounds induce apoptosisin cancer cells.

The triterpene compounds of this invention can reduce blood vessel inthe tumor in a subject. (FIG. 44)

The following results are obtained from MicroArray experiments: Y/D isthe ratio (in folds) of gene expression in cells treated with compound Yas compared with those of the no drug control (D), YM/D is the ratio ofgene expression in cells treated with compound YM (ACH-Y, Y withoutsugar moiety) compared with those of the no drug control (D)

TABLE 1 Effect of Y and YM on fibronectin expression in ES2 cells ProbeSet ID Y/D YM/D Gene.Symbol Gene.Title 212464_s_at −2.7 −1.1 FN1fibronectin 1 216442_x_at −2.6 −1.1 FN1 fibronectin 1 211719_x_at −2.6−1.2 FN1 fibronectin 1 210495_x_at −2.5 −1.1 FN1 fibronectin 1

The results of the microarray experiment showed that compound Y andYM(ACH-Y) inhibit fibronectin expression; The expression ratio ofcompound Y/Y3 to the control are −2.7, −2.6, −2.6, −2.5 folds detectedby gene probes 212464_s_at; 216442_x_at; 211719x_at and 210495_x_at,respectively. These results indicate Y/Y3 inhibits fibronectinexpression; wherein the YM/ACH-Y also show minor fibronectin inhibitionwith the inhibiting ratio of −1.1, −1.1, −1.2, −1.1 folds by gene probes212464_s_at; 216442_x_at; 211719_x_at and 210495_x_at, respectively. Theresults indicate that while YM is active but is less potent than

TABLE 2 Effects of Y and YM on integrin (vitronectin receptor)expression in ES2 cells Probe Set ID Y/D YM/D Gene.Symbol Gene.Title202351_at −1.8 −1.3 ITGAV integrin, alpha V (vitronectin receptor, alphapolypeptide, antigen CD51) 236251_at −1.4 −1.4 ITGAV Integrin, alpha V(vitronectin receptor, alpha polypeptide, antigen CD51)

The results of the micro array experiment showed that compound Y andYM(ACH-Y) inhibit integrin (vitronectin receptor) expression; whereinthe inhibiting ratio of compound Y/Y3 to the control are −1.8, −1.4,folds as detected by different probes; wherein the inhibiting ratio ofYM (ACH-Y) to the control are −1.3, −1.4 folds.

TABLE 3 Effects of Y and YM on laminin expression in ES2 cells Probe SetID Y/D YM/D Gene.Symbol Gene.Title 202202_s_at −2.2 −2.0 LAMA4 laminin,alpha 4 216264_s_at −2.0 −2.0 LAMB2 laminin, alpha 5 200770_s_at −1.9−1.1 LAMC1 laminin, alpha 6 211651_s_at −1.6 −1.7 LAMB1 laminin, alpha 7201505_at −1.6 −2.0 LAMB1 laminin, beta 1

The results of the micro array experiment showed that compound Y andYM(ACH-Y) inhibit laminin expression; The expression ratio of compoundY/Y3 to the control are −2.2, −2.0, −1.9, −1.6, −1.6 folds as detectedby different probes; wherein the inhibiting ratio of YM/ACH-Y to thecontrol are −2.0, −2.0, 1.1, −1.7, −2.0 folds.

TABLE 4 Effects of Y and YM on CAM expression in ES2 cells Probe Set IDY/D YM/D Gene.Symbol Gene.Title 201952_at −1.9 −1.4 ALCAM activatedleukocyte cell adhesion molecule 201951_at −1.9 −1.7 ALCAM activatedleukocyte cell adhesion molecule 212425_at −1.7 −1.5 SCAMPI Secretorycarrier membrane protein 1 240655_at −1.6 −1.3 ALCAM Activated leukocytecell adhesion molecule 212417_at −1.4 −1.4 SCAMP1 secretory carriermembrane protein 1 239431_at −1.3 −1.3 TICAM2 toll-like receptor adaptormolecule 2 212416_at −1.3 −1.1 SCAMP1 secretory carrier membrane protein1 228234_at −1.3 −1.3 TICAM2 toll-like receptor adaptor molecule 2206667_s_at −1.3 −1.5 SCAMP1 secretory carrier membrane protein 1

The micro array experiment showed that compound Y and YM(ACH-Y) inhibitgene expression related to the adhesion molecule; wherein the inhibitingratio of compound Y/Y3 to the control are −1.3 to −1.9 folds as detectedby different probes; wherein the inhibiting ration of YM/ACH-Y to thecontrol are −1.1 to −1.7 folds.

TABLE 5 Effects of Y and YM on collagen expression in ES2 cells ProbeSet ID Y/D YM/D Gene.Symbol Gene.Title 217428_s_at −3.0 −1.2 COL10A1collagen, type X, alpha 1 (Schmid metaphyseal chondrodysplasia)231766_s_at −2.8 −2.4 COL12A1 collagen, type XII, alpha 1 201438_at −2.4−1.5 COL6A3 collagen, type VI, alpha 3 1556138_a_at −2.2 −2.8 COL5A1Collagen, type V, alpha 1 211809_x_at −2.0 −1.5 COL13A1 collagen, typeXIII, alpha 1 207543_s_at −2.0 −1.5 P4HA1 procollagen-proline, 2-oxoglutarate 4-dioxy- genase (proline 4-hydro- xylase), alpha poly-peptide I 213992_at −2.0 −1.9 COL4A6 collagen, type IV, alpha 6211343_s_at −1.9 −1.7 COL13A1 collagen, type XIII, alpha 1 211966_at−1.8 −1.7 COL4A2 collagen, type IV, alpha 2 200656_s_at −1.8 −1.2 P4HBprocollagen-proline, 2- oxoglutarate 4-dioxy- genase (proline 4-hydro-xylase), beta polypeptide (protein disulfide iso- merase-associated 1)209081_s_at −1.7 −1.5 COL18A1 collagen, type XVIII, alpha 1 202619_s_at−1.7 −1.2 PLOD2 procollagen-lysine, 2- oxoglutarate 5-dioxy- genase 2203325_s_at −1.7 −2.8 COL5A1 collagen, type V, alpha 1 200827_at −1.7−1.2 PLOD1 procollagen-lysine 1, 2- oxoglutarate 5-dioxy- genase 1221730_at −1.6 −1.6 COL5A2 collagen, type V, alpha 2 202311_s_at −1.6−3.6 COL1A1 collagen, type I, alpha 1 213110_s_at −1.6 −2.2 COL4A5collagen, type IV, alpha 5 (Alport syndrome) 212091_s_at −1.6 −1.9COL6A1 collagen, type VI, alpha 1 213290_at −1.6 −1.5 COL6A2 collagen,type VI, alpha 2 211981_at −1.6 −2.2 COL4A1 collagen, type IV, alpha 1200654_at −1.6 −1.3 P4HB procollagen-proline, 2- oxoglutarate 4-dioxy-genase (proline 4-hy- droxylase), beta poly- peptide (protein disulfideisomerase-associated 1) 212489_at −1.5 −4.1 COL5A1 collagen, type V,alpha 1 202620_s_at −1.4 −1.3 PLOD2 procollagen-lysine, 2- oxoglutarate5-dioxy- genase 2 202733_at −1.4 −1.9 P4HA2 procollagen-proline, 2-oxoglutarate 4-dioxy- genase (proline 4-hy- droxylase), alphapolypeptide II 208535_x_at −1.4 −1.2 COL13A1 collagen, type XIII, alpha1 202185_at −1.3 −1.1 PLOD3 procollagen-lysine, 2- oxoglutarate 5-dioxy-genase 3 202465_at −1.3 −1.6 PCOLCE procollagen C-endo- peptidaseenhancer 221729_at −1.3 −1.8 COL5A2 collagen, type V, alpha 2242324_x_at −1.3 −1.8 CCBE1 collagen and calcium binding EGF domains 11568611_at −1.3 −2.4 P4HA2 Procollagen-proline, 2- oxoglutarate 4-dioxy-genase (proline 4-hydro- xylase), alpha poly- peptide II

The results of the micro array experiment showed that compound Y andYM(ACH-Y) inhibit collagen expression. The expression ratio of compoundY/Y3 to the control range from −1.3 to −3.0 folds; wherein theexpression ratio of YM/ACH-Y to the control range from −1.1 to −3.6folds

TABLE 6 Effects of Y and YM on integrin expression in ES2 cells ProbeSet ID Y/D YM/D Gene.Symbol Gene.Title 205422_s_at −1.9 −2.0 ITGBL1integrin, beta-like 1 (with EGF-like repeat domains) 202351_at −1.8 −1.3ITGAV integrin, alpha V (vitro- nectin receptor, alpha polypeptide,antigen CD51) 1557080_s_at −1.7 −2.5 ITGBL1 Integrin, beta-like 1 (withEGF-like repeat domains) 214927_at −1.7 −1.8 ITGBL1 Integrin, beta-like1 (with EGF-like repeat domains) 205885_s_at −1.7 −2.0 ITGA4 integrin,alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) 213416_at−1.6 −1.7 ITGA4 integrin, alpha 4 (antigen CD49D, alpha 4 subunit ofVLA-4 receptor) 215177_s_at −1.6 1.1 ITGA6 integrin, alpha 6 205884_at−1.6 −1.7 ITGA4 integrin, alpha 4 (antigen CD49D, alpha 4 subunit ofVLA-4 receptor) 1555349_a_at −1.6 −1.4 ITGB2 integrin, beta 2 (antigenCD18 (p95), lymphocyte function-associated antigen 1; macrophage antigen1 (mac-1) beta subunit) 227259_at −1.6 −1.1 CD47 CD47 antigen (Rh-related antigen, integrin- associated signal transducer) 201474_s_at−1.6 −1.5 ITGA3 integrin, alpha 3 (antigen CD49C, alpha 3 subunit ofVLA-3 receptor) 214021_x_at −1.5 −2.2 ITGB5 Integrin, beta 5 201656_at−1.5 −1.1 ITGA6 integrin, alpha 6

The results of the micro array experiment showed that compound Y andYM(ACH-Y) inhibit gene expression related to the integrin family in ES2cells. The expression ratio of compound Y/Y3 to the control are rangingfrom −1.5 to −1.9 folds; wherein the expression ratio of YM/ACH-Y to thecontrol are ranging from −1.1to −2.5 folds.

TABLE 7 Effects of Y and YM on myosin expression in ES2 cells Probe SetID Y/D YM/D Gene.Symbol Gene.Title 211926_s_at −2.2 −1.2 MYH9 myosin,heavy poly- peptide 9, non-muscle 212372_at −1.7 −1.4 MYH10 myosin,heavy poly- peptide 10, non-muscle 212338_at −1.7 −2.1 MYO1D myosin ID204527_at −1.6 −1.2 MYO5A myosin VA (heavy poly- peptide 12, myoxin)202555_s_at −1.6 −1.2 MYLK myosin, light polypeptide kinase /// myosin,light poly- peptide kinase 203215_s_at −1.6 −1.6 MYO6 myosin VI225080_at −1.5 −1.4 MYO1C Myosin IC 224823_at −1.5 −1.4 MYLK myosin,light poly- peptide kinase

The results of the micro array experiment showed that compound Y andYM(ACH-Y) inhibit gene expression related to the myosin family in ES2cells. The expression ratio of compound Y/Y3 to the control are rangingfrom −1.5 to −2.2 folds; wherein the expression ratio of YM/ACH-Y to thecontrol are ranging from −1.2 to −2.1 folds.

TABLE 8 Effects of Y and YM on cadherins expression in ES2 cells ProbeSet ID Y/D YM/D Gene.Symbol Gene.Title 244091_at −2.0 −1.7 CDH13Cadherin 13, H-cadherin (heart) 202468_s_at −1.9 −1.6 CTNNAL1 catenin(cadherin-associated protein), alpha-like 1 204726_at −1.8 −1.7 CDH13cadherin 13, H-cadherin (heart) 207149_at −1.7 −2.2 CDH12 cadherin 12,type 2 (N- cadherin 2) 201533_at −1.5 −1.2 CTNNB1 catenin(cadherin-associated protein), beta 1, 88 kDa 204677_at −1.5 −1.1 CDH5cadherin 5, type 2, VE- cadherin (vascular epithelium) 208407_s_at −1.5−1.7 CTNND1 catenin (cadherin-associated protein), delta 1 203440_at−1.4 −1.0 CDH2 cadherin 2, type 1, N- cadherin (neuronal) 210844_x_at−1.4 −1.2 CTNNA1 catenin (cadherin-associated protein), alpha 1, 102 kDa

The results of the micro array experiment showed that compound Y andYM(ACH-Y) inhibit gene expression of cadherins family in ES2 cells.

TABLE 9 Effects of Y and YM on tenascin-C expression in ES2 cells ProbeSet ID Y/D YM/D Gene.Symbol Gene.Title 201645_at −3.2 1.0 TNC Tenascin C(hexabrachion)

The results of the micro array experiment showed that compound Y inhibitgene expression of cadherins family in ES2 cells.

TABLE 10 Effects of Y and YM on heparin sulfate expression in ES2 cellsProbe Set ID Y/D YM/D Gene.Symbol Gene.Title 201655_s_at −2.4 −1.3 HSPG2heparan sulfate proteoglycan 2 (perlecan) 219403_s_at −1.4 −1.3 HPSEHeparanase 203284_s_at −1.3 −1.7 HS2ST1 heparan sulfate 2-O-sulfotransferase 1

The results of the micro array experiment showed that compound Y inhibitgene expression of heparin sulfate family in ES2 cells.

TABLE 11 Effects of Y and YM on CD54 expression in ES2 cells Probe SetID Y/D YM/D Gene.Symbol Gene.Title 202638_s_at 1.6 2.3 ICAM1intercellular adhesion molecule 1 (CD54), human rhinovirus receptor

The results of the micro array experiment showed that compound Ystimulate gene expression of CD54 in ES2 cells.

TABLE 12 Effects of Y and YM on angiopoietin expression in ES2 cellsProbe Set ID Y/D YM/D Gene.Symbol Gene.Title 205572_at 3.5 1.4 ANGPT2angiopoietin 2 211148_s_at 2.5 1.4 ANGPT2 angiopoietin 2 205609_at −1.1−1.2 ANGPT1 angiopoietin 1 221009_s_at −1.2 −1.4 ANGPTL4angiopoietin-like 4 227533_at −1.5 −2.3 ANGPTL1 Angiopoietin-like 1

The results of the micro array experiment showed that compound Y andYM(ACH-Y) modulate the gene expression of angiopoietin family in ES2cells. There is a up regulation of (positive regulating on) angiopoietin2 and a down regulation of (negative regulating on) angiopoietin 1 andangiopoietin-like 1 and angiopoietin-like 4.

TABLE 13 Effects of Y and YM on Glypican expression in ES2 cells ProbeSet ID Y/D Ym/D Gene.Symbol Gene.Title 243865_x_at −2.7 −2.0 GPC6Glypican 6 227059_at −2.2 −1.8 GPC6 Glypican 6

The results of the micro array experiment showed that compound Y and Yminhibit gene expression of Glypican in ES2 cells.

TABLE 14 Effects of Y and YM on regulator of G-protein expression in ES2cells Probe Set ID Y/D Ym/D Gene.Symbol Gene.Title 204339_s_at −3.5 −1.2RGS4 regulator of G-protein signalling 4 204337_at −3.4 −1.1 RGS4regulator of G-protein signalling 4 204338_s_at −2.5 −1.1 RGS4 regulatorof G-protein signalling 4

The results of the micro array experiment showed that compound Y and YMinhibit gene expression of G-protein in ES2 cells

TABLE 15 Effects of Y and YM on thrombospondin in ES2 cells Probe Set IDY/D Ym/D Gene.Symbol Gene.Title 201109_s_at −2.0 −6.2 THBS1thrombospondin 1 201110_s_at −1.8 −4.5 THBS1 thrombospondin 1201108_s_at −1.7 −2.2 THBS1 thrombospondin 1

The results of the micro array experiment showed that compound Y and YMinhibit gene expression of thrombospondin in ES2 cells

TABLE 16 Effects of Y and YM on insulin-like growth factor bindingprotein expression in ES2 cells Probe Set ID Y/D YM/D Gene.SymbolGene.Title 210095_s_at −4.0 −3.5 IGFBP3 insulin-like growth factorbinding protein 3 212143_s_at −3.7 −5.3 IGFBP3 insulin-like growthfactor binding protein3 201508_at −1.7 −2.4 IGFBP4 insulin-like growthfactor binding protein 4 205302_at −1.7 −1.8 IGFBP1 insulin-like growthfactor binding protein 1 201163_s_at −1.4 −2.7 IGFBP7 insulin-likegrowth factor binding protein 7 203851_at −1.3 −1.8 IGFBP6 insulin-likegrowth factor binding protein 6

The results of the micro array experiment showed that compound Y and YMinhibit gene expression of insulin-like growth factor binding protein inES2 cells.

TABLE 17 Effects of Y and YM on RAB3B, member RAS oncogene familyprotein expression in ES2 cells ID Y/D YM/D Gene.Symbol Gene.Title242629_at −3.5 −1.8 RAB3B RAB3B, member RAS oncogene family 205924_at−1.7 −1.8 RAB3B RAB3B, member RAS oncogene family 227123_at −1.6 −1.2RAB3B RAB3B, member RAS oncogene family 205925_s_at −1.2 −1.2 RAB3BRAB3B, member RAS oncogene family

The results of the micro array experiment showed that compound Y and Yminhibit gene expression of RAB3B, member RAS oncogene family protein inES2 cells.

TABLE 18 Effects of Y and YM on potassium channel, subfamily U, proteinexpression in ES2 cells ID Y/D YM/D Gene.Symbol Gene.Title 237273_ at−4.0 −1.9 KCNU1 potassium channel, subfamily U, member 1

The results of the micro array experiment showed that compound Y and Yminhibit gene expression of family protein relate to potassium channel inES2 cells.

TABLE 19 Effects of Y and YM on phosphatase, protein expression in ES2cells 37028_at 2.4 5.6 PPP1R15A protein phosphatase 1, regulatory(inhibitor) subunit 15A 202014_at 2.6 6.2 PPP1R15A protein phosphatase1, regulatory (inhibitor) subunit 15A 215501_s_at 3.2 7.1 DUSP10 dualspecificity phosphatase 10 221563_at 3.8 5.7 DUSP10 dual specificityphosphatase 10

The results of the micro array experiment showed that compound Y and Ymstimulate gene expression of family protein relate to phosphatase.

(Results of F1 and F3) In these experiments, we established anddescribed the basic phenomenon that Y-treatment of ES2 cancer cellscause inhibition of fibronectin secretion. With a Western blot assay, weshowed that ES2 cells without drug treatment (DMSO control) secretFibronectin to medium and the amount of Fibronectin accumulated withtime. However, no or only minimally secretion of Fibronectin wasobserved in cell culture treated with Xanifolia-Y. Inhibition ofFibronectin was observed as early as 8 hours after drug-treatment.

Inhibition of Fibronectin secretion is physiological and thedetermination of its quantity is based on the following criteria:

-   -   1. Fibronectin is secreted from viable cells. Only cell with        over 85% viable cells after drug-treatment are employed in these        experiments. The viable cells were determined by MTT assay.    -   2. For comparison, the immuno-band intensity from each sample        are normalized with cell mass. The cell mass was determined by        the MTT assay and is assigned as an MTT unit for each cell        sample.

(Results of F4) Under a sub-lethal drug concentration (10 ug/ml Y), Over95% of cells after 18 hours of Y-treatment was viable as determined byMTT assay. Western Blots show a reduction of Fibronectin secretion bycells into culture medium after Y-treatment. Scan of Fibronectin Westernbands (average 6 pairs of blots) shows that there is a 40% reduction ofFibronectin secretion after 18 hours of Y-treatment.

(Results of F5) Similarly, 85% of cells after 24 hours of Y-treatmentwere viable as determined by the MTT assay. Western blot shows areduction of Fibronectin bands of Y-treated samples. Based on 6 pairs ofblots and after normalize them to MTT units, a 31% reduction ofFibronectin band intensity of Y-treated samples was observed.Accordingly, these results indicate that Fibronectin secretion by cellsreduce 31% after 24 hours of Y-treatment.

(Results of F7) Effects of Paclitaxel on Fibronectin secretion by ES2cells. To demonstrate that not all anticancer drugs can inhibitFibronectin secretion from cells, we employed Paclitaxel, a well-knownanticancer drug that is effective for ovarian cancer. Our results showedthat there is no inhibition of Fibronectin secretion with Paclitaxeltreatment in ES2 cells (10 to 50 ng/ml, the IC50 of Paclitaxel is 1.5ng/ml). This study also showed that Fibronectin secreted by ES2 cellsreduced 30-40% after Y-treatment which agrees with previous results.

(Results of F8) In addition to ES2 cells, another human ovarian cancercells (Hey8A) were employed in this study. It was found that Y-treatedHey8A cells secrete 31% Fibronectin as compared with the DMSO control,accordingly it has a inhibition of 69%.

Beside ovarian cancer, other human cancer cells were tested in thefollowing experiments. These experiments show that the secretion ofFibronectin from cancer cells derived from lung, bladder, liver, brainand skin is inhibited by Xanifolia-Y treatment.

(F11) For lung carcinoma cells (H460), at concentration of 20 ug/ml,there are inhibitions of Fibronectin secretion ranged from 20-60%.

(F12A) For bladder carcinoma cells (HTB-9), Xanifolia-Y (10 ug/ml)inhibits 50% of Fibronectin secretion.

(F15) In liver HepG2 cells. 10 ug/ml of xanifolia-Y inhibits 42%secretion of Fibronectin.

(F16) Incubation of brain glioblastoma T98G cells with 10 ug/ml ofxanifolia-Y inhibits 27% Fibronectin secretion and with 20 ug/ml Yinhibits 74% Fibronectin secretion.

(F17) For skin SK-Mel-5 cells, the inhibition is 40-57% with 20 ug/ml ofXanifolia-Y.

Studies of Xanifolia-Y Analogs and Other Saponin on FibronectinSecretion from ES2 Cells.

(F 23) To study the inhibition effect with other saponins, we tested thecompound 054, a triterpenoid saponin isolated from the same plant. With054, there is no inhibition activity of Fibronectin secretion in ES2cells, even at higher dose of 40 ug/ml (instead of the usual effectiveconcentration of 10 ug/ml). This result indicates there is specificityin triterpenoid saponin that is responsible for the inhibition effect.

(F21) To research for functional groups that are effective forFibronectin inhibition activity, we tested several derivatives ofxanifolia-Y3. In these experiments, ES2 cells treated with ACH-Y (Y3without sugars) and AKOH-Y (Y3 without the C21, C22 angeloyl group).

With 20 ug/ml Ach-Y, there is a reduction of Fibronectin secretion fromES2 cells (ranging from 53%-75% of the control).

Inhibition of Fibronectin secretion was less (or not observed) when only10 ug/ml Ach-Y was used. However, no effect was observed with AKOH evenat 80 ug/ml.

(F 13) We also tested for inhibition activity with beta-Escin, atriterpenoid saponin with only one angeloyl group attached at C21.

The results show that 10 and 20 ug/ml of beta-Escin inhibit 7% and 48%,respectively, of Fibronectin secretion from ES2 cells. But 10 ug/ml ofxanifolia-Y inhibits 49% Fibronectin secretion. Results indicate thatbeta-Escin also inhibits Fibronectin secretion but has half potency asxanifolia-Y.

(F14) (F24) We have determined the inhibition effect of differentanalogs of xanifolia-Y on ES2 cells. The results are shown in thefollowing table.

ES2 cells β- ES- X- Y0- Y1- Y3- Y7- ACH- AKOH- 10 10 10 10 10 10 Y-20 80% 19 39 34 41 47 34 48 No inhibition effect

All samples (except AKOH) tested have effects of inhibition ofFibronectin secretion from ES2 cells. With 80 ug/ml of AKOH-Y which is 4times higher concentration used in others saponins (10 ug/ml), it stillhas no effect on inhibition of Fibronectin secretion on ES2 cells.

In conclusion, saponins in general have effects in inhibition ofFibronectin secretion from ES2 cells. The fact that AKOH-Y (the Y3without diangeloyl group) does not show any activity, indicating thatacylation of C21, 22 positions is important for the inhibition activity.

Studies of Other Saponins on Fibronectin Secretion from ES2 and OtherCells

ES2 cells Mb12- Mb5- ACH- Ba1- Ba4- AKOH- 10 10 Mb5-10 10 10 Mb-40 %inhibition 30 35 30 30 28 No effectLiver

HepG2 Mb12-10 Mb5-10 ACH-Mb5-10 Bal-10 Ba4-10 % inhibition 30 35 25 3328Lung

H460 Mb12-10 Mb5- 10 ACH-Mb5-10 Bal-10 Ba4-10 % inhibition 20 25 22 1918Bladder

HTB-9 Mb12-10 Mb5-10 ACH-Mb5-10 Bal-10 Ba4-10 % inhibition 32 28 30 2530Brain

T98G Mb12-10 Mb5-10 ACH-Mb5-10 Bal-10 Ba4-10 % inhibition 40 33 35 26 24Skin

SK-MEL-5 Mb12-10 Mb5-10 ACH-Mb5-10 Bal-10 Ba4-10 % inhibition 17 15 2010 10

In addition to ES2 cells, other cancer cells derived from differentorgans were also investigated. Results are shown in following tables.

(F25, 26, 31B) Liver

HepG2 β- ES- X- Y0- Y1- Y3- Y7- ACH- 10 10 10 10 10 10 Y-30 % 44 42 4033 48 10 21 inhibition

(F27,29) Lung

H460 β-ES- X- Y0- Y1- Y3- Y7- ACH- 20 20 10 10 10 10 Y-20 % No 37 22 1319 18 28 inhibition effect

(F28, 30) Bladder

HTB-9 β- ES- X- Y0- Y1- Y3- Y7- ACH- 10 10 10 10 10 10 Y-30 % 47 38 3250 51 60 No inhibition effect

F 31, 32) Brain

T98G β- ES- X- Y0- Y1- Y3- Y7- ACH- 20 20 10 10 10 10 Y-20 % 66 52 22 4026 24 30 inhibition

(F 33) Skin

SK-MEL-5 β- ES- X- Y0- Y1- Y3- Y7- ACH- 20 20 10 10 10 10 Y-30 % 17 1527 10 11 No 21 inhibition effect

(F20) Determination of cellular contents and secretion of Fibronectinafter xanifolia-Y-treatment

Results: This experiment shows that (1) there is a 46% reduction (54% ofcontrol) of Fibronectin secretion after xanifolia-Y-treatment and (2)the Fibronectin cellular content decrease 70% (30% of control) after theY-treatment; (3) there is no change of the cellular beta-actin contentin ES2 cells after the Y-treatment.

Up Regulation of Angiopoietin 2 (Ang2) in ES2 Cells with Xanifolia-YTreatment.

Methods: ES2 (human ovarian carcinoma cells) were grew in RPMI 1640medium. 4.5 million cells were seeded in a T75 flask and grown for 24hours before drug-treatment.

Drug-treatment: Cells cultures were treated with 5, 10 and 15 ug/ml(final concentration) of Xanifolia-Y3 [Y3-5, Y3-10, Y3-15]. or DMSOcontrol [D-10]. After 24 hours, cells were suspended in 1 ml of SDSsample buffer (cell-extract). Samples (80 ul/lane) were applied to 10%SDS gel and electrophoresis was conducted with 100 volts for 2 hours.Protein was transferred to a nitrocellulose membraneelectrophoretically. The nitrocellulose blot was blocked with 5% non-fatdry milk in PBS. The blot was then incubated with the first antibodies(goat anti-Ang2, SIGMA A0851) and second antibody (donkey anti-goat APconjugated, Promega V115A). The immuno-bands were developed withBCIP/NBT color development system (Promega S3771).

Results: As shown in FIG. 43, a Angiopoietin-2 immuno-band (M.W. 66K)was observed in cell extract from cells treated with 15 ug/mlXanifolia-Y. No detectable or minimal immuno-band of Angiopoietin-2 wasobserved in control and low concentration of xanifolia-Y under theseconditions. These results indicate that treatment of Xanifolia-Y in ES2cells increase the cellular content of Angiopoietin-2. These resultscorroborate the results of Microarray studies.

This invention provides compositions and methods for modulating the geneexpression in cancer cells, wherein the modulating comprises of positiveand negative regulation, wherein genes being modulated are adhesionproteins; wherein modulation includes expression, production andsecretion of adhesion proteins, wherein the adhesion proteins comprisefibronectin, integrins family, Myosin, vitronectin, collagen, laminin,cadherin, heparin, tenascin, CD 54, CAM. This invention providescompositions and methods for modulating angiopoietins, wherein comprisespositive regulating the angiopoietin 2, wherein comprises negativeregulating angiopoietin 1. The composition and method of this inventioncomprises a triterpene wherein acylation group at carbon position 21and/or 22 of the triterpene is necessary for the function and areselected from angeloyl, acetyl, alkanoyl, alkenoyl and acyl group. Thesugar moiety (ies) at position 5 of the triterpene is important forenhancing activity of these compounds.

EXPERIMENTAL DETAILS

Experiment details of herb extraction, analysis of extract components byHPLC, determination of the cell-growth activity effected by Xanifolia Ywith cells derived from different human organs using MTT Assay,purification of the bioactive components from plant extract,fractionation of plant extracts with FPLC, isolation of component Yswith preparative HPLC, determination of the chemical structure isdisclosed in PCT/US05/31900, U.S. Ser. No. 11/289,142.

Experiment 1: Determination of the Hemolytic Activities of Compound Yfrom Xanthoceras sorbifolia

Methods:

-   -   Human whole blood was obtained from the Houston Gulf Coast Blood        Center.    -   Red blood cells were isolated by the following method: Human        blood (in EDTA) was diluted 1:1 with PBS, underlay with 4 mL of        Histopaque-1077 (SIGMA) and was centrifuged at 400 g for 30 min.    -   Red blood cells (RBC) were collected and washed three times with        PBS.    -   10% suspensions of RBC were prepared with PBS before use.    -   50 μL of RBC suspension was added to 2 mL of saponins with        different concentration.    -   The suspension was mixed by vortexing then left to sit at room        temperature for 60 minutes.    -   The suspension was centrifuged at 3000 rpm for 5 min. Absorbance        of the supernatant was measured at 540 nm.        Results:

In this experiment, hemolytic activities of human red blood cells byXanifolia-Y (#63Y), Escin and SIGMA saponin standard were compared. Ycontains two angeloyl groups, Escin has one angeloyl group and SIGMAsaponin standard is a mixture of saponins from Quillaia bark. Theresults show that #63Y (compound Y) has higher hemolytic activity(IC50=1 μg/mL) than Escin or SIGMA saponin standard (IC50=5 μg/mL). SeeFIG. 5, Panel A.

Experiment 2: Determination the Hemolytic and MTT Activities of CompoundY after Removal of the Angeloly Group or the Sugar Moiety by Alkaline orAcid Hydrolysis, Respectively

Methods:

(A) Alkaline Hydrolysis of Xanifolia-Y: 20 mg of Xanifolia-Y wasdissolved in 0.5 mL of 1M NaOH. The solution was incubated in an 80° C.water bath for 4 hours. It was cooled to room temperature before beingneutralized with 0.5 mL 1 N HCl (adjusted pH to about 3). The mixturewas extracted with 2 mL 1-butanol 3 times. The butanol fractions werecollected and lyophilized. The hydrolyzed saponin was further purifiedwith HPLC in a C-18 column eluted with 25% acetonitrile.

(B) Acid Hydrolysis of Xanifolia-Y: 15 mg Xanifolia-Y was dissolved in 1mL of Methanol. 1 mL of 2N HCl was then added. The mixture was refluxedin an 80° C. water bath for 5 hours. The solution was then neutralizedby adding 2 mL of 1N NaOH (to a final pH 3-4). The aglycone was thenextracted with ethylacetate 3 mL×3. The extracts were collected andpooled. Further isolation of aglycone (sugar-removed Xanifolia-Y) wasachieved by HPLC with isocratic elution of 80% acetonitrile.

Results:

The angeloly groups or the sugar moiety of the compound Y were removedby alkaline or acid hydrolysis respectively. The hemolytic activities ofthe hydrolysed products were then analyzed. Results of these studiesindicate that removing sugars from the compound Y reduced hemolyticactivity, but removing the angeloyl groups from the compound Y destroyedthe hemolytic activity. It also suggested that sugars are helpful butnot essential for hemolytic activity. See FIG. 3, Panel D. Theexperiment results show that compound-Y lost MTT activities if theangeloyl groups were removed. However, the MTT activities became veryweak when the sugar moiety of the compound was removed. See FIG. 4,Panels C and D. Results of comparison of hemoyltic activities betweenCompound Y, Escin from SIGMA are shown in FIG. 6. Results of thecomparison of hemolytic activities between compound Y, compound Ywithout sugar moiety or angeloly groups are shown in FIG. 5, Panels Aand B. Chemical structures of compound Y without sugar moiety (ACH-Y) orangeloly groups (AKOH-Y) are shown in FIG. 6 respectively.

Experiment 3: Effects of Xanifolia-Y on Reduction of VenousInsufficiency, Particularly Hemorrhoids

Methods:

SD rats, male, age-matched 7-8 weeks old weighing 163±18 g were in theexperiment. The tested animals were allowed to acclimate for a week.

A cotton swab with a diameter of 4 mm soaked with 0.16 mL of inducer(deionized water:pyridine:ethyl ether:6% croton oil/ethyl ether,1:4:5:10) was applied to the rat's anus for 12 seconds. The finalconcentration of croton oil was 3%. The edema developed linearly until7-8 hours after application and the severity of the edema was sustainedfor more than 24 hours. Twenty-four hours later, recto-anus tissue(approx. 10 mm long) was isolated after the rats were euthanized. Theweights of rat body and recto-anus were measured. The recto-anuscoefficient (RAC) was calculated using the formula: weight of recto-anus(mg)/body weight (g).RAC=weight of recto-anus (mg)/body weight (g)×100%

The rates were randomly divided into 5 groups: control, positive controland 3 test groups, and each group had 8 rats. The dose for 3 groups ofrats are 10, 20 and 40 mg/kg. The dose of 0.5% CMC-Na for control groupis the same as each test group. The tested drug was fed into the stomachon the morning, once a day before the animal modeling for 5 days. Theanus suppository was applied to positive control once after the animalmodeling (1 mL/100 g). The weights of recto-anus and RACs werecalculated, compared with the controls and subject to a student t-test.

Results

The edema formed 30 minutes after the treatment. The rats wereeuthanized 22 hours after the last administration. The results showedthat Xanifolia Y significantly reduced the swelling of the recto-anus ofrats (Table A1).

TABLE A1 Effects of Xanifolia Y on reduction of swelling of therecto-anus of rats Weight of recto-anus Reduction Group (g) RAC rate %Modeling 6.20 ± 0.77 2.33 ± 0.36 10 mg/Kg 4.68 ± 0.77* 1.83 ± 0.36* 21.520 mg/Kg 4.28 ± 0.60** 1.61 ± 0.24** 30.9 40 mg/Kg 3.97 ± 0.65** 1.51 ±0.23** 35.1 Anus 3.90 ± 0.80** 1.54 ± 0.36** 33.9 suppository n = 8, X ±SD, *p < 0.05, **p < 0.01

Experiment 4: Effects of Xanifolia Y on Reduction of the Swelling ofRats' Feet in the Carrageenin-Induced Swollen Feet Model in Rats

Method:

SD rats, male, weighing 163±18 g, were used in the experiment. Thetested animals are allowed to acclimate for a week. The rats drink waterfreely. The rats were randomly divided into 5 groups: control, positivecontrol and 3 test groups, and each group had 8 rats. The doses for 3groups of rats are 10, 20 and 40 mg/kg. Indometacin for the positivecontrol is 10 mg/kg and fed once after modeling. The dose of CMC-Na forthe control group is the same as each test group and fed into thestomach once a day before modeling for 5 days. The tested drug was fedinto the stomach 10 minutes before the animal modeling (1 mL/100 g). Thevolumes of right foot of each rat were measured 0.5, 1, 2 and 4 hoursbefore and after modeling. The volumes of the right hind feet weremeasured at a different time, 10 minutes after inflammation induced bysubcutaneous injection (with syringe needle 7) of 0.05 mL of the 1% ofCarrageenin/normal saline mixture into the feet. The Rate of swelling ofthe feet was calculated, compared with the controls and subject to astudent t-test.Rate of swelling (E) (%)=L _(tn) −L _(t0) /L _(t0)×100%

-   -   L_(tn): volume of foot after the inflammation    -   L_(t0): volume of foot before the inflammation        Results:

The results of this experiment showed Xanifolia Y significantly reducedswelling of the feet and the effect was related to the dosages. (TableA2).

TABLE A2 Effects of Xanifolia Y on reduction of swelling of rats' feetinduced by Carrageenin Swelling rate after administration (%) GroupDosage(mg/kg) 0.5 hour 1 hour 2 hours 4 hours Modeling 22.6 ± 8.1 27.6 ±8.2 23.0 ± 10.1 12.9 ± 6.1 Test 10 16.7 ± 3.8 18.5 ± 6.2 16.0 ± 5.9 10.5± 7.2 Test 20 10.5 ± 4.1** 13.6 ± 4.2** 12.4 ± 5.3*  8.5 ± 5.4 Test 4010.3 ± 3.3** 12.6 ± 4.7** 12.5 ± 6.2*  6.5 ± 5.4 Indometacin 10.0 11.8 ±4.3** 14.7 ± 6.5* 12.8 ± 7.0** 10.7 ± 8.8 n = 8, X ± SD, *p < 0.05, **p< 0.01 Student-t

Experiment 5: Purification of the Inhibition Components in theXanthoceras Sorbifolia Extract

(A) Fractionation of Plant Extracts with FPLC

Methods

Column. Octadecyl functionalized silica gel. Column dimension: 2 cm×28cm; equilibrated with 10% acetonitrile—0.005% TFA before use.

Sample loading: 1-2 ml, concentration: 100 mg/ml in 10%acetonitrile/TFA.

Gradient elution condition: 10-80% acetonitrile in a total volume of 500ml.

Monitor absorption wavelength: at 254 nm.

Fraction Collector: 5 ml/fractions (collect from 10% to 72%acetonitrile)

Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.

Results

The elution profile of the chromatography shows 4-5 broad fractions.These fractions were analyzed with HPLC. Specific components,corresponding to a-z in these FPLC fractions. FPLC fractions are thengrouped into 7 pools and analyzed for cell growth activity with MTTassay. The fractions contain inhibition activity can be found.

(See PCT/US05/31900, filed Sep. 7, 2006; U.S. Ser. No. 10/906,303, filedFeb. 14, 2005; International Application No. PCT/US04/43465, filed Dec.23, 2004; International Application No. PCT/US04/33359, filed Oct. 8,2004 and U.S. Ser. No. 11/131,551, filed May 17, 2005, the contents ofwhich are incorporated herein by reference).

(B) Isolation of Component Ys with Preparative HPLC Methods

Column: A preparative HPLC column (Waters Delta Pak C18-300A); Elutionconditions: 45% acetonitrile isocratic elution with flow rate of 1ml/min.

Fractions are monitored at 207 nm and were collected and lyophilized.

Results

Final separation of Y fractions was achieved by HPLC with a preparativecolumn. These fractions, which include compound Y0, Y1, Y2, Y or Y3 andY4, were collected. Re-chromatography of compound Y showed a single peakin HPLC with a C18 reverse phase column. Re-chromatography of thecompound Y8, Y9 and Y10 showed a single peak in HPLC with a C18 reversephase column.

(C) Appearance and Solubility

The pure compound Ys is an amorphous white powder, soluble in aqueousalcohol, i.e., methanol or ethanol, 50% acetonitrile and 100% pyridine.

(D) Inhibition Analysis of Compound Ys with MTT Assay

Inhibition analysis of compound Y was determined with MTT assay. FIG. 4Ashows the inhibition activities of compound Y, Y8, Y9 and Y10 on thegrowth of ovarian cancer cells (OCAR-3). FIG. 22 shows the inhibitionactivities of compound Y(Y3), Y0, Y1 and Y2 on the growth of ovariancancer cells (OCAR-3)

Experiment 6: Determination of the Chemical Structure Methods

NMR analysis. The pure compound Y of Xanthoceras sorbifolia wasdissolved in pyridine-D5 with 0.05% v/v TMS. All NMR spectra wereacquired using a Bruker Avance 600 MHz NMR spectrometer with a QXI probe(1H/13C/15N/31P) at 298 K. The numbers of scans for 1D 1H spectra were16 to 128, depending on the sample concentration. 2D HMQC spectra wererecorded with spectral widths of 6000×24,000 Hz and data points of2024×256 for t2 and t1 dimensions, respectively. The number of scans was4 to 128. 2D HMBC were acquired with spectral widths of 6000×30,000 Hzand data points of 2024×512 for t2 and t1 dimensions, respectively. Thenumber of scans was 64. The 2D data were zero-filled in t1 dimension todouble the data points, multiplied by cosine-square-bell windowfunctions in both t1 and t2 dimensions, and Fourier-transformed usingsoftware XWIN-NMR. The final real matrix sizes of these 2D spectra are2048×256 and 2048×512 data points (F2×F1) for HMQC and HMBC,respectively.

Mass spectral analysis. The mass of samples was analyzed by (A)MALDI-TOF Mass Spectrometry and by (B) ESI-MS Mass spectrometry. (A)Samples for MALDI-TOF were first dissolved in acetonitrile, and thenmixed with the matrix CHCA, i.e., Alpha-cyano-4-hydroxycinnamic acid, 10mg CHCA/mL in 50:50 water/acetonitrile and 0.1% TFA in finalconcentration. The molecular weight was determined by high resolutionmass spectroscope analysis with standards. (B) For ESI, the sample wasanalyzed with LCQ DECA XP Plus machine made by Thermo Finnigan. It isionized with ESI source and the solvent for the compound isacetonitrile.

Results

The profile of the proton NMR is presented in (application Ser. No.11/683,198, US20070161580, filed Mar. 7, 2007) FIGS. 8A, 8B, 9A, 9B,10A, 10B and. The NMR profiles of 1H, 13C, TOCSY, HMQC, HMBC and NOESYare shown respectively. FIG. 11 shows the MS of Y0. Based on these dataand analysis, the structure of compound Y0 is assigned as shown below.

Structure of Compound Y0

This invention provides a bioactive compound Y0 and the chemical nameis:

3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-angeloyl, 22-O-(2-methylpropanoyl)-3β,15α, 16α, 21β, 22α, 28-hexahydroxyolean-12-ene,

Experiment 7: Animal Study

Methods

-   -   Athymic Nu/Nu mice are divided into three groups (A, B and C)        with four animals in each group.    -   On day 0, mice of group A and B were transplanted        intra-peritoneally with ES2 (human ovarian cancer) cells.    -   On day 1, mice from B and C groups received drug (Xanifolia-Y,        by i.p. route at dose of 5 mg/kg)    -   On days 2 to 4, and 7 to 11, B and C groups animals received        daily drug administration of Xanifolia-Y, by i.p. route at dose        of 2.5 mg/kg.    -   Group A mice have no drug-treatment.

Results:

Group A Mice—Died on day 19-22

Group B Mice—Survived over 50 days

Group C Mice—Survived over 50 days

-   -   Also See FIG. 23

Experiment 8: Animal Study

Methods

-   -   Athymic Nu/Nu mice were divided into three groups (A, D and E)        with four animals in each group.    -   On day 0, all mice were transplanted intra-peritoneally with ES2        (human ovarian cancer) cells.    -   Group A mice received no drug-treatment.    -   Group D: From day 4, mice received a daily drug administration        of Xanifolia-Y, via i.p. route for 9 days at dose of 2.5 mg/kg.    -   Group E: From day 10, mice received daily drug administration of        Xanifolia-Y, via i.p. route for 10 days at dose of 2.5 mg/kg.        Result:

Group A, Mice implanted with tumor and no drug. All died within 24 days

Group D, Mice implanted with tumor and were given drug 9 times from 4thday. All survived

Group E Mice implanted with tumor and were given drug 10 times from 10thday. Half the number of mice survived

Also See FIG. 24

Experiment 9: Animal Study

-   -   Athymic Nu/Nu mice (2-3 months old) were transplanted sc with        ES2 (human ovarian cancer) cells.    -   Five days after the transplant (day one), mice were divided into        two groups (H and J) with two animals in each group.    -   Group H: On days 1-5, and 8-10 mice received daily drug        administration of Xanifolia-Y, by i.p. route at dose of 2.5        mg/kg.    -   Group J mice received no drug-treatment.        Result:

Group H: Mice received drug-treatment, tumor size is 10 mm in 10 days

Group J: Mice received no drug-treatment, tumor size is 18 mm in 10 days

The tumor size is 45% smaller in mice with drug than the mice with nodrug

-   -   in 10-day period.        See FIG. 25

Experiment 10: Aminal Study

Methods

-   -   Athymic Nu/Nu mice (5-6 weeks old) are divided into three groups        (0, P and Q) with 5-6 animals in each group.    -   On day 0, all mice were transplanted intra-peritoneally with ES2        (human ovarian cancer) cells.    -   Group 0: animals received no drug-treatment.    -   Group P: On days 4-8, 11-15, 18-22, 25-29, 32-36, 39-43, animals        received daily drug administration of Xanifolia-Y, by i.p. route        at dosage of 2.5 mg/kg    -   Group Q: On days 10-15, 18-22, 25-29, 32-36, 39-43, animals        received daily drug administration of Xanifolia-Y, by i.p. route        at dosage of 2.5 mg/kg.

The median survival time of tumor bearing mice with drug-treatmentstarting on day 4 after tumor inoculation is 58 days (extension of lifespan of 141%); and the median survival time of tumor bearing mice withdrug-treatment started on day 10 after tumor inoculation is 31 days(extension of life span of 29%). See FIG. 30

Experiment 11: Studies of Effect on Human Ovarian Cancer Cell Lines

Since we found that ovarian carcinoma cell lines are among the sensitivecells studied, we further investigated if other ovarian carcinoma celllines are also susceptible to Xanifolia-Y.

Majority of ovarian cancers arise from the surface epithelium of ovary,most of them belong to the histological subtypes of clear cell andserous carcinoma. We obtained 10 more human ovarian carcinoma cell-linesof these histological subtypes for these studies. The inhibitionactivity exerted by Xanifolia-Y was determined with MTT assay. Thefollowing table shows the IC50 values of Xanifolia-Y on these celllines.

Results:

TABLE B2 IC50 values of human ovarian carcinoma determined by MTT assay.Cell lines Types IC50 (uM) OVCAR3 Serous 2.2 TOV-21G Clear cell 2.2 ES2Clear cell 4.4 RMG2 Clear cell 8.8 OVCA 429 Serous 7 OVCA 432 Serous 4.4OVCA 433 Serous 8.8 Caov 3 Serous 7 SKOV 3 Serous 10.5 Hey 8A Serous10.5

The IC50 values of Xanifolia-Y in these cell-lines are ranging from 2 to10 uM. These studies show that the effective concentration ofXanifolia-Y is in the micro-molar range which is comparable to those ofother anti-cancer drugs.

Experiment 12: Study Apoptosis Induced by Xanifolia-Y

Experiment of apoptosis of OVCAR3 cells after treatment with Xanifolia-Ywas assessed with flow cytometry of GFP-Annexin-V and propidium iodide.

Results were shown in FIG. 33 which indicates that induction of theearly apoptosis (the lower right quadrant) and late a poptotic/necrosis(the upper right quadrant) were found in cells 24 h after exposure toXanifolia-Y. By comparing the distribution of apoptotic/necrotic cellsafter the drug-treatment, a higher number of early apoptotic cells wereobserved as compare to those of the late apoptotic/necrosis cells. Theseresults indicate that apoptosis is a major form of cell death induced byXanifolia-Y. See FIG. 26.

Experiment 13: Effect of Xanifolia-Y on Membrane Structure (EM Studies)

Xanifolia-Y has a potent hemolytic activity in red blood cells (FIG.27). To study the effect of Xanifolia-Y on membrane structure, themorphology of cell membrane treated with Xanifolia-Y was examined withEM. In this experiment, K562 cells were treated with 5 uM of Xanifolia-Yfor 60 min. Solvent DMSO and AKOH-Y (a derivative of Xanifolia-Y withoutthe angeloyl group and it has no activity) served as controls. Cellswere negative stained with 1% UAc and subsequently examined with EM.

FIG. 27 show that patches of pits were found in the membrane ofXanifolia-Y treated cells (FIG. 27B) but not in cells treated with theDMSO (FIG. 27A) or AKOH-Y (FIG. 27C) controls. These pits have the sizefrom 80 A to 500 A (in diameter). The pits represent holes formed in themembrane. The pits are arranged in a characteristic pattern with smallerpits (80 A in diameter) located in the periphery and the bigger ones(500 A in diameter) in the center. The bigger holes are resulted fromfusing of the smaller holes (FIG. 27D).

Membrane image of cells treated with A: DMSO solvent control, 60 min(magnification: ×60,000); B: Xanifolia-Y 5 uM, 60 min. (×60000); C:AKOH-Y, 20 uM, 60 min. (×60000); D: Xanifolia-Y 5 uM, 60 min. (×20000)

This experiments results show that the Xanifolia-Y alters the membraneof cell.

See FIG. 27

Experiment 14: Inhibition of Cell Adhesion by Xanifolia-Y

Methods and Results. ES2 or Hey8A cells were plated in T25 flasks withmedium containing 5 ug/ml of Xanifolia-Y. Cultures were incubated for 5hours. Attached cells were removed from flasks by trypsinization and theamounts were counted. Compare to no drug controls, 86±4% of ES2 cellsand 67±8% of Hey8A cells were found attached to flasks under thiscondition. At 5 ug/ml Xanifolia-Y, over 90% of unattached cells arealive as determined by the trypan Blue exclusion assay and by theirability to re-attach to flasks when plating in medium withoutXanifolia-Y. However, with 10 ug/ml Xanifolia-Y, less than 40% of cellsattached to flasks and many of them are dead cells. This experimentshows that Xanifolia-Y inhibits cells adhesion process.

Experiment 15: Combined Inhibition Effect of Xanifolia-Y and Paclitaxel

Methods: ES2 cells were exposed to (i) Xanifolia-Y with concentrationsof 40, 20, 10, 5, 2.5, and 1.25 ug/ml; or (ii) Paclitaxel withconcentrations of 10, 5, 2.5, 1.25, 0.62 and 0.031 ng/ml; or (iii)Combined Xanifolia-Y and Paclitaxel with concentrations of each drug insame order (for example, 40 ug/ml Y plus 10 ng/ml T; 20 ug/ml Y plus 5ng/ml T, etc. please see FIG. 28). Cells growth under these conditionswas determined by the MTT assay.

Results: As shown in FIG. 28, The IC50 for Xanifolia-Y and Paclitaxel,is 5 ug/ml and 1.25 ng/ml, respectively. Additive effect was observedwhen both drugs were used, because in this case, the IC50 value forPaclitaxel (0.625 ng/ml) and for Xanifolia-Y (2.5 ug/ml) is less thanthose when they are used singly.

See FIG. 28.

Experiment 16: Identify the Binding Target of Xanifolia-Y of AdhesionProteins and Signaling Proteins in Ovarian Cancer Cells

In our animal studies, it was shown that Xanifolia-Y extended the lifespan of tumor bearing mice (FIGS. 23 and 24). The animals died sooner ifthe treatment of Xanifolia-Y was delayed (comparing results oftreatments started from 1, 4 or 10 days after tumor inoculation). Theresults show that Xanifolia-Y inhibits migration or metastasis of theinoculated cancer cells. Ovarian carcinoma cells express high levels ofadhesion molecules. Adhesion proteins are present in both cancer cellsand mesothelial cells. While the lost of adhesion is blocking of theprotein accessibility due to direct binding to Xanifolia-Y, theinteraction of Xanifolia-Y with membrane alter indirectly the adhesionprotein's binding site(s).

A. We label Xanifolia-Y and use it as ligand to find the targetmolecules bind to it. Xanifolia-Y has carbohydrates (Galactose,arabinose and glucoronic acids). These carbohydrates can be ³H-labeledwith peridodate-tritiated Borohydride (Cahmberg and Andersson, 1977).For the control, the non-reactive derivative of Xanifolia-Y (AKOH-Y)also is labeled. Labeled Xanifolia-Y is purified by HPLC and verifiedthat it retains activity before use. Our studies indicate that thecarbohydrates of Xanifolia-Y do not contribute a major role in itsactivity. Therefore, ³H-labeling of carbohydrate in Xanifolia-Y shouldnot affect its activity.

B. We employ the following methods to study the direct binding betweenthe labeled Xanifolia-Y and its target.

I. Detect binding of Xanifolia-Y on cell surface (by autoradiography):This autoradiographic method employs labeled Xanifolia-Y adding toculture cells (K562 suspension cells, or ES2 monolayer cells) anddetermine cells pick up label on cells surface. Cells are incubated inmedium with labeled Xanifolia-Y with a concentration equal to the IC50value (e.g. 4.4 ug/ml for ES2 cells). At this concentration, cells arealive. After incubation (30 min to 1 hour), cells are washed, fixed,dried and emulsified. Radioactive Xanifolia-Y is detected withautoradiography and microscopic analysis. Radioactive AKOH-Y (notactive) serves as a negative control. This experiment shows thatXanifolia-Y binds to membrane.

II. Determine binding between selected protein and Xanifolia-Y by RIA:This experiment determines the known adhesion proteins bind toXanifolia-Y. Binding of Xanifolia-Y to known purified adhesion proteins(or other signaling proteins) can be determined with RIA (Radio-ImmunoAssay). Antibodies of many of these adhesion proteins (and proteinitself) are available and can be employed in RIA assay. Binding ofradioactive Xanifolia-Y (which compete with non-radioactive Xanifolia-Y)with specific protein is immuno-precipitated by specific antibodies.Affinity binding constant for these known proteins is determined by thismethod.

III. Immuno-precipitation (IP) analysis: Xanifolia-Y binds to moleculesthat associated with adhesion proteins. Many of known signaling proteinsor adhesion protein form complex (spheroids). Spheroids areimmuno-precipitated with the specific antibody to one of the adhesionprotein. Cells are cultured with radioactive Xanifolia-Y. Membraneproteins or spheroids are isolated. Alternatively, isolated spheroidsfrom cells are incubated with radioactive Xanifolia-Y in a cell-freesystem. Co-IP of the radioactive Xanifolia-Y in the spheroid indicatesits association of Xanifolia-Y-binding protein. After the spheroid isidentified, the contents of the spheroid are analyzed with biochemicalmethods, such as 2D gel electrophoresis.

IV. Purification and identification of Xanifolia-Y-binding protein: Weemploy the radioactive Xanifolia-Y as a tracer to identify and purifyits target. The effective concentration for Xanifolia-Y in vitro assayis in the micro-molar range, suggesting that the binding to its targetis relatively tight (or with high affinity). Therefore, it does able todetect its binding target in cell-free system. Membrane is isolated andproteins will be fractionated with ion-exchanger and gel filtrationchromatography. Fractions from chromatography will be analyzed (byradioactive counting) for their association of Xanifolia-Y.Alternatively, proteins isolated from cancer cells or the membranefraction are separated by 2D gel, blot the proteins in NC membrane andincubate with radioactive labeled Xanifolia-Y and determine the targetprotein that pick up radioactive Xanifolia-Y (tagged by thelabeled-Xanifolia-Y). The tagged target is detected by autoradiography(beta-imager). The identity of protein is determined by Peptide Mappingand MALDI-TOF technique.

Our compounds interacting with target protein comprise integrins family,CD44, fibronectin, Myosin VI, collagen, laminin, Glycosylation cellsurface proteins, polyglycans and FAK Adhesive molecules play animportant role in migration and metastasis of ovarian cancer (Skubitz,2002, Schaller, 1996; Zetter, 1993). A major route for the spread ofovarian cancer is by the attachment of tumor cells to the mesotheliumlining in the peritoneal cavity (Gardner et al., 1995). For example,serous ovarian cancer cells invade through their membrane and releasedproteolytic enzymes or EMC molecules for attachment to mesothelial cells(Skubitz, 2002).

The integrins family and CD44—These proteins are detected on allmesothelial cells and ovarian cancer cells and play an important role intumor/mesothelial interaction (Gardner et al., 1995).

Ovarian carcinoma cells form multicellular spheroids, in the peritonealcavity of patients with advanced disease (Burleson et al., 2004). It wasshown that adhesive proteins in ovarian carcinoma multicellularspheroids were involved in the adhesion process (Casey et al., 2001).The adhesive proteins are: integrins, CD44 and fibronectin.

Myosin VI, a motor protein that regulates border cell migration, isabundantly expressed in high-grade ovarian carcinomas including ES2cells. Yoshida et al., (2004) reported that inhibition of myosin VIexpression in ES2 cells impeded ovarian cells spreading and migration.

Casey et al., (2003) reported that Glycosylation cell surface proteinsand polyglycans mediate the adhesion, migration and invasion of ovariancarcinoma cells. The major ligand for CD44 is the extra-cellular matrixglycosaminoglycan and hyaluronic acid (HA). Mesothelial cells containlarge amount of HA (Catterall et al., 1997). These surface proteins alsoinclude: fibronectin, collagen and laminin.

FAK (focal-adhesion-associated kinase). FAK is a protein tyrosine kinasewhich involves in the regulation of cell cycle progression, cellsurvival and cell migration (Schaller, 2001). It was reported that FAKpromote cell motility and invasion of ovarian cancer through distinctsignaling pathway (Hsia et al., 2003). The role of integrins foradhesion is to activate intracellular signaling pathways (Schaller andParsons, 1993). One of the affected kinase is (FAK). FAK expressioninhibited by Xanifolia-Y-treatment.

Experiment 17: The Effect of Xanifolia-Y in Preventing Nodule Formationin Peritoneal Cavity

This experiment shows Xanifolia-Y blocks migration, invasion or growthof ovarian cancer in peritoneal cavity.

Our animal experiments indicate that the life span of the tumor bearingmice is extended after the Xanifolia-Y-treatment. The results show thata sooner treatment provides a better protection. The results show thatXanifolia-Y inhibits tumor cell's growth and reduce their attachments tomesothelium linings. We use Hey8A cells inoculated in peritoneal cavityproduce tumor nodules (solid tumor mass) and the numbers of nodulesincrease during the tumor progression (Lander Jr., et al., 2005).

The numbers of tumor nodules are determined by laparotomy. The growth ofnodules (number and mass) before and after Xanifolia-Y-treatment isdetermined. The results show that Xanifolia-Y inhibits the growth numberof nodules; and inhibits tumor growth after the Xanifolia-Y-treatment.

(A) Animal Model: Hey8A Cells are Inoculated into Peritoneal Cavity.

The relationship between the nodule formation and timelines during tumorprogression are established. Usually, 4 to 6 nodules are formed duringthe entire period of tumor progression in this system (Lander Jr., etal., 2005). We start the drug-treatment at times equivalent to 20% and50% of full tumor progression. At these times, the number of nodules isless and the size (weight) of the nodules is smaller. At the end of thedrug-treatment, animals are euthanized and number of tumor nodules isdetermined and the tumor size (mass) is weighted.

Animals: 40 nu/nu mice are used per experiment.

2 groups of mice (20 each) (i) Drug-treatment start at time equivalentto 20% of full tumor progression (or on 4 days after tumor inoculation)(ii) Drug-treatment starts at time equivalent to 50% of full tumorprogression (or on 10 days after tumor inoculation). On the day ofstarting drug-treatment, half of the animals (10) will be scarified andthe number and weight of nodules will be measured.

Drug-Treatments:

Animals are administered daily for 5 days per week for two-three weeks.Dose: 0.25 mg/kg, through i.p. route.

Result:

The numbers of tumor nodules are less in mice with drug treatmentcompare to mice without drug treatment. The size of tumor in drugtreatment mice is smaller than tumor in no-drug treatment mice.

(B) Animal Model: ES2 Cells are Inoculated into Peritoneal Cavity.

The relationship between the nodule formation and timelines during tumorprogression are established. Usually, 4 to 6 nodules are formed duringthe entire period of tumor progression in this system (Lander Jr., etal., 2005). We start the drug-treatment at times equivalent to 20% and50% of full tumor progression. At these times, the number of nodules isless and the size (weight) of the nodules is smaller. At the end of thedrug-treatment, animals are euthanized and number of tumor nodules isdetermined and the tumor size (mass) is weighted.

Animals: 40 nu/nu mice are used per experiment.

2 groups of mice (20 each) (i) Drug-treatment start at time equivalentto 20% of full tumor progression (or on 4 days after tumor inoculation)(ii) Drug-treatment starts at time equivalent to 50% of full tumorprogression (or on 10 days after tumor inoculation). On the day ofstarting drug-treatment, half of the animals (10) will be scarified andthe number and weight of nodules will be measured.

Drug-Treatments:

Animals are administered daily for 5 days per week for two-three weeks.Dose: 0.25 mg/kg, through i.p. route.

Result:

The numbers of tumor nodules are less in mice with drug treatmentcompare to mice without drug treatment. The size of tumor in drugtreatment mice is smaller than tumor in no-drug treatment mice.

We use two histological types of human ovarian cancer ES2 and Hey8A.These two are most common ovarian cancer types: clear cells carcinoma(ES2) and serous carcinoma (Hey8A). Hey and ES2 show high expression ofintegrins and other adhesion proteins (Ahmed et al., 2005).

Ovarian cancer cells that are resistant to drugs are chosen in thisstudy. ES2 cells express low levels of P-glycoprotein and havemulti-drug chemotherapy resistant character (resistant to doxorubicin,cisplatin, carmustine, etoposide and cyanomorpholinodoxorubicin).

Experiment 18: Determination of Aquaporin in HeLa and OVCAR3 Cells

Methods:

1. Hela or OVCAR3 cells were cultured in RPMI 1640 medium at 37 C in anincubator with 5% C02.

2. Cells were harvested, washed with PBS.

3. Cellular protein was dissolved in SDS sample buffer with proteaseinhibitors (PMSF and Leupeptin) and was incubated at 70 C for 20 minbefore use.

4. Equal amounts of protein from Hela or OVCAR3 cells were separatedwith 12% SDS gel and subsequently blotted on nitrocellulose paper.

5. Western blot was performed with anti-AQ1 antibody (Chemicon/SIGMA)and second antibody which was conjugated with Alkaline phosphatase.

Results:

The following figure shows the results of Western blot.

Aquaporin-1 (indicated with an arrow) was observed in OVCAR3 cells butwas minimally detected in HeLa cells.

Based on same amounts of protein loading into gel, it was found thatOVCAR3 cells have higher concentration of Aquaporin-1 than in Helacells.

Since OVCAR3 cells are more sensitive to Xanifolia-Y and it has a higherconcentration of Aquaporin-1, these results show Xanifolia-Y is potentto inhibit the cancer cell growth wherein the Aquaporin isoverexpressed.

See FIG. 31

Experiment 19

Analysis of Gene Expression of ES2 Cells after Y-Treatment by Microarray

In this invention, the microarray experiments were done in studying thegene expression. Total number of 54676 genes has been studied.

Cell culture and drug-treatment: ES2 cells were seeded in a T-25 flaskwith 4.5 million cells per flask for 24 hours. Cell culture was replacedwith fresh medium with xanifolia-Y (Y) or DMSO no drug control (D) for24 hours. Cells were then harvested for RNA isolation. Three experimentswere done.

RNA extraction, labeling, hybridization, and data analysis. RNA wasextracted from tumor cells using the Qiagen RNeasy Kit. RNA quality andquantity were checked by the Agilent BioAnalyzer and the NanoDrop®ND-1000 spectrophotometer respectively before further manipulation. Thefirst and second cDNA strands were synthesized from 20 ng of total RNAusing the Affymetrix T7 oligo(dT) primer protocol and kit for thetwo-cycle amplification. To produce amplified biotin-labeled-cRNA, thecDNA was reverse transcribed by in vitro transcription using theMegaScript kit from Ambion. 15.0 μg of the labeled cRNA was fragmentedand re-checked for concentration using the NanoDrop® ND-1000spectrophotometer. A hybridization cocktail containing Affymetrixspike-in controls and fragmented labeled cRNA was loaded onto the HumanU133 Plus 2.0 GeneChip® oligonucleotide array. The Affymetrix array(Affymetrix, Inc. Santa Clara, Calif.) is comprised of over 1,300,000unique oligonucleotide features that represent greater than 38,500well-substantiated human genes. The array was hybridized for 16 hours at45° C. with rotation at 60 rpm then washed and stained with astrepavidin, R-phycoerythrin conjugate stain on the Affymetrix FluidicisStation 450. Signal amplification was done using biotinylatedantistreptavidin. The arrays were scanned using the GeneChip® 3000confocal laser scanner with autoloader. The images were analyzed andquality control metrics recorded using Affymetrix GCOS software version1.4. Lastly, the expression value for each gene was calculated usingdChip PM-only model based or Plier algorithm.

Data Analysis Methods

Pairwise comparisons were made as follows: Treated vs. Control (Y vs.D), Modified Drug vs Control (YM/ACY-H vs. D) and Treated vs. ModifiedDrug (Y vs. YM/ACH-Y)

Cel files analyzed using the Bioconductor package of R Statisticalprogramming. Limma analysis generated a reasonable number of changinggenes between the samples.

The raw data in the .CEL files were normalized by the GCRMA method(robust multi-array analysis). It is implemented in Bioconductor(http://www.bioconductor.org/). The raw signal intensity data werenormalized, background corrected and summarized based on certainstatistical models, and an expression value, in log 2-scale, is obtainedper chip per probe set. Then the null hypothesis was tested that there'sno significant changes in gene expression between the treatment pairs.This was done by LIMMA and is also implemented in Bioconductor. It usesempirical Bayes method to estimate the variance in gene expression. Onecomparison was made, namely, High Grade vs. Low Grade. The raw p-valueswere adjusted by the Benjamnin-Hochberg method for false discovery rate(FDR) control. All data sets contained a significant number of geneswith a p-value less than 0.05, which is that the probability that a geneis NOT differential expressed (false positive) is 1:20.

All expression data is filtered by p-value (0.05).

The raw p-values were adjusted by the Benjamnin-Hochberg method forfalse discovery rate (FDR) control to yield an adjusted p-value.

Results: Please see Table 1 to 12

Inhibition of Fibronectin Secretion by Xanifolia-Y (Western Blot)

Experiment 20 (F1)

Methods:

Cells: ES2 cells were grown in T-25 flask with RPMI 1640 mediumovernight before drug treatment. Drug treatment: cell cultures werereplaced with fresh RPMI medium with Xanifolia-Y (10 ug/ml finalconcentration) or DMSO (as control) at 0 hour. At 1, 2, 4, 8 and 24hours, aliquot of culture medium was taken out for Fibronectindetermination. Fibronectin was determined by Western blot withmonoclonal antibody (SIGMA) specific to human Fibronectin only.

Results (also see FIG. 39):

-   -   1. Cells treated with DMSO (as no drug control) secret        Fibronectin to medium and the amount of Fibronectin accumulated        with time. There is no or only minimally secretion of        Fibronectin observed in cell culture treated with Xanifolia-Y.    -   2. For controls, Fibronectin immunoband was not observed in RPMI        medium with fetal bovine serum, or employing the normal mouse        serum (NS1).

Experiment 21(F3)

Methods:

Cells: ES2 cells were grew in RPMI 1640 medium over night beforedrug-treatment. Drug-treatment: cells cultures were replaced with freshRPMI medium containing Xanifolia-Y (10 ug/ml final concentration) orDMSO (as control) at 0 hour. At 4 hours (A) or 8 hour (B), culturemedium was replaced with fresh culture medium without drug. At 2, 4, 8and 24 hours, aliquot of culture medium was taken out for Fibronectindetermination. Fibronectin (FN) was determined by Western blot withmonoclonal antibody (SIGMA) specific only to human Fibronectin.

Results (also see FIG. 40):

(1) Compare the control and Y-treated cells before drug removal (at 4and 8 hours), there is a reduction of FN secretion from Y-treated cells.There is no obvious cell morphology change during these times,suggesting cells are alive.

(2) Compare the control and Y-treated cells after the removal of drug at24 hours, it was estimated that secretion of FN from Y-treated cells wasreduced to over 50%.

(3) The amount of FN secreted by Y-treated cells at 24 hours is higherthan those at 8 hours (before removal of Y) indicating that cells arestill alive after Y-treatment.

Experiment 22 (F4)

Methods:

Cells: ES2 cells were grew in RPMI 1640 medium over night beforedrug-treatment. Drug-treatment: cells cultures were replaced with freshRPMI medium containing Xanifolia-Y (10 ug/ml final concentration) orDMSO (as control) at 0 hour. At 2 and 18 hours, aliquot of culturemedium was taken out for Fibronectin determination (Western blotmethod). Cell viability at 18 hours was determined by MTT assay.Cultures were replaced with RPMI medium with MTT and incubated for anhour. The formation of formazan was dissolved in DMSO and OD at 570 nmwas measured.

Results (also see FIG. 40):

-   -   Over 95% of cells after 18 hours of Y-treatment were viable as        determined by MTT assay.    -   Western Blots show a reduction of FN secretion by cells into        culture medium after Y-treatment. Scan of FN Western bands        (average 5 sets of blots) shows that there is a 40% reduction of        FN secretion after 18 hours of Y-treatment.

Experiment 23(F5)

Methods:

Cells and Drug-treatment: same as previous experiments. After 7 or 24hours, aliquot of culture medium was taken out for Fibronectindetermination (Western blot method). Cell viability at 24 hours wasdetermined by MTT assay.

Results:

-   -   93% and 85% of cells after 7 and 24 hours, respectively, of        Y-treated cells was viable as determined by MTT assay.    -   Change in Fibronectin secretion during the first 7 hours of        Y-treatment is not noticeable. However, after 24 hours, as        compared with the control, the Fibronectin band of Y-treated        samples is reduced. Based on same amount of live cells, the        intensity of the immuno-bands were compared (per MTT O.D. unit).        The scan of 3 pairs of blots shows a 31% reduction of        Fibronectin band. Accordingly, these results indicate that        Fibronectin secretion by cells reduce 31% after 24 hours of        Y-treatment.

Experiment 24(F 7): Effects of Paclitaxel on Fibronectin Secretion byES2 Cells

Methods:

Cells: ES2 cells were grew in RPMI 1640 medium over night beforedrug-treatment. Drug-treatment: cells cultures were replaced with freshRPMI medium containing DMSO (as control) [D]; Xanifolia-Y (10 ug/ml)[Y]; or Paclitaxel 10 or 50 ng/ml [T10, ro T50]. After 24 hours, aliquotof culture medium was taken out for Fibronectin determination (Westernblot method). Cell viability at 24 hours was determined by MTT assay.

Results:

Based on the MTT units (cell basis) of treated cells and compared thosewith the DMSO control, 87%, 94% and 91% growth of Y, T10 and T50 cells,respectively, were viable after 24 hours of treatment.

The amount of Fibronectin secreted by cells into medium was determinedby Western blot assay. The amount of Fibronectin secreted per cellsbasis was determined by dividing the Western-band intensity with the MTTunit.

By comparing with the DMSO control, ES2 cells treated with 10 ng/ml or50 ng/ml Taxel secret 105% or 97%, respectively, of Fibronectin intomedium during 24 hours of treatment. At the same time, Y-treated ES2cells secreted 62% of control (a reduction of 38%).

Experiment 25(F 8): Hey8A Cells Treated with Xanifolia-Y

Methods:

Cells: Hey8A (human ovarian carcinoma cells) were grew in RPMI 1640medium to 90% confluent before drug-treatment. Drug-treatment: Cellscultures were replaced with fresh RPMI medium containing either DMSO (ascontrol) [D1]; or Xanifolia-Y (10, 15, or 20 ug/ml) [Y1, Y2 and Y3].Aliquot of medium was removed as 0 hours sample and no FN was detectedat this time. After 24 hours, aliquot of culture medium was taken outfor Fibronectin determination (Western blot method). Cell viability at24 hours was determined by MTT assay. Cultures were replaced with RPMImedium (5 ml) with MTT and incubated for an hour. The formation offormazan was dissolved in DMSO and OD at 570 nm was measured.

Western Blot: Spent culture medium (0.6 ml) was mixed with SDS samplebuffer (0.2 ml), boiled for 3 minutes before loading to SDS gel. Samples(60 ul/lane) were applied to a 6% SDS gel and electrophoresis wasconducted with 100 volts for 2 hours. Protein was transferred to anitrocellulose paper electrophoretically (30 min at 100 volts). TheWestern blot was incubated with the first antibody (mouse anti-FN,specific to human FN, SIGMA F0916) and second antibody (Anti-mouse IgGAP conjugated, Promega S3721). The immunobands were developed withBCIP/NBT color development system (Promega S3771).

Results:

-   -   After 24 hours of drug-treatment, cells with 15 ug/ml and 20        ug/ml were found dead (floating) and were not further proceeded.        Cells with DMSO and 10 ug/ml Y were processed.    -   The MTT assay showed that the growths of cells with Y-treatment        (10 ug/ml) are 83% of control.    -   The Western blot show that the band intensity of Y-treated        samples (Y1) is much reduced compare to the DMSO control (D1)    -   The average band intensity after corrected with the MTT unit        are: 1179 and 366, for DMSO control and Y-treated samples,        respectively. Accordingly, Y-treated Hey8A cells secrete 31%        Fibronectin (FN) as compared with the DMSO control, or a 69%        inhibition.

Experiment 26(F 11): Inhibition of Fibronectin Secretion by Xanifolia-Yin Human Lung Carcinoma Cells (H460)

Methods: please see Experiment 20.

Results: Lung cells (H460) are sensitive to Y in inhibition of FNsecretion. Based on MTT results, cells are still viable at 20 ug/ml Y,but the inhibition of FN is over 60%.

Experiment 27(F 12): Inhibition of Fibronectin Secretion by Xanifolia-Yin Bladder Carcinoma Cells (HTB-9)

Methods: please see Experiment 20.

Results:

-   -   The MTT assay showed that the growth of cells treated with 10        ug/ml Y reduced to 77%-91% compared to the DMSO control.    -   The Western blot shows that the FN band intensity of Y-treated        samples are reduced. After corrected with the MTT unit        (equivalent to cell mass) there is about 50% reduction of FN        band intensity per cell mass.    -   These results indicate that Xanifolia-Y (10 ug/ml) inhibit 50%        of FN secretion.

Experiment 28(F 13): ES2 Cells Treated with Y and Beta-Escin

Methods: Please See Experiment 20

Results:

-   -   The MTT assay showed that the growth of cells with Y, Es10 and        Es20 are 89%, 90% and 82%, respectively as compared to the DMSO        control.    -   The Western blot show that the FN band intensity of Es10 and        Es20 samples are 93% and 52%, respectively, of DMSO control. The        band intensity of Y10 sample is 51% of control.    -   These results show that 10 and 20 ug/ml of beta-escin inhibit 7%        and 48%, respectively, of FN secretion. But 10 ug/ml of Y        inhibits 49% FN secretion.    -   Results indicate that beta-escin also inhibits FN secretion but        with half potency as Xanifolia-Y.

Experiment 29(F 14): ES2 Cells Treated with Different Xanifolia-Ys

Methods: Please See Experiment 20

-   -   Two experiments were done (FN14B and FN14C). Five gels per each        experiment were run.        Results:

Except for AKOH-Y (the Y3 without diangeloyl group), all samples havesome degrees of inhibition of FN secretion from ES2 cells. 80 ug/ml ofAKOH-Y which is 4 times higher concentration used in others saponins (10ug/ml), still have no effect on inhibition of FN secretion on ES2 cells.

ES2 cells β- ES- X- Y1- Y3- Y7- AKOH- 10 10 10 10 10 80 % 19 39 41 47 34No inhibition effectConclusion:

It seems that saponins in general have effects in inhibition ofFibronectin secretion from ES2 cells. However, this experiment foundthat acylation of C21, 22 positions is important for the inhibitionactivity.

Experiment 30(F 23): ES2 Cells Treated with 054

Methods: Please see Experiment 20.

Results: Based on these results, there is no inhibition of FN secretionin ES2 cells with 054 treatment at 40 ug/ml.

Experiment 31(F 24): ES2 Treated with Y0 and Y5

Methods: please see Experiment 20.

Results: By comparing the immuno band's intensities of these samples theresults of this experiment indicate that: (1) Y5 has same activity as Y3for inhibition of FN secretion (both inhibit 68% at 10 ug/ml);

(2) Y0 is weaker as compare to Y3 for inhibition of FN secretion.(inhibit 34% at 10 ug/ml);

(3) Conclusion, both Y0 and Y5 have inhibition activity for FN secretionfrom ES2 cells.

Experiment 32(F 25, 26): HepG2 Cells Treated with Ys

Methods: Please see Experiment 20.

Results: By comparing the immuno band's intensities of these samples(see table), it was found that at concentration of 10 ug/ml. X, ES, Y0,Y1, Y3, and Y5 have inhibition effect on Fibronectin secretion fromHepG2 cells. Minimum or No effect was observed with Y7, Ach (10 ug/ml)and AKOH (80 ug/ml).

HepG2 β-ES- X- Y0- Y1- Y3- Y7- ACH- 10 10 10 10 10 10 Y-30 % 44 42 40 3348 10 21 inhibition

Experiment 33(F 27, 29): NCI-H460 Cells (Lung) Treated with Ys

Methods: Please See Experiment 20

H460 β-ES- X- YO- Y1- Y3- Y7- ACH- 20 20 10 10 10 10 Y-20 % No 37 22 1319 18 28 inhibition effect

Experiment 16 (F 28, 30): HTB-9 Cells (Bladder) Treated with Ys

Methods: Please See Experiment 20

(FN28, 30) Bladder

HTB-9 β-ES- X- Y0- Y1- Y3- Y7- ACH- 10 10 10 10 10 10 Y-30 % 47 38 32 5051 60 No inhibition effect

Experiment 34 (F31, 32): T98G (Brain) Treated with Y2

Methods: Please See Experiment 20

TG98G Y0-10 Y1-10 Y3-10 Y7-10 X-20 ES-20 ACH-20 % 22 40 26 24 52 66 30inhibition

Experiment 18(F 33): SK-MEL-5 Cells Treated with Ys

Methods: Please See Experiment 20

SK-MEL-5 β-ES- X- Y0- Y1- Y3- Y7- ACH- 20 20 10 10 10 10 Y-30 % 17 15 2710 11 No 21 inhibition effect

Experiment 35(F 20): Determination of Cellular Contents and Secretion ofFN after Y3-Treatment

Methods:

Cells: ES2 (human ovarian carcinoma cells) were grew in RPMI 1640medium. 1.5 million cells were seeded in a T25 flask and grown for 24hours before drug-treatment. Drug-treatment: Cells cultures werereplaced with fresh RPMI medium containing either 2.5 ul of DMSO (ascontrol) [D]; or 10 ug/ml (final concentration) of Xanifolia-Y3 [Y].After 24 hours, aliquot of culture medium was taken out for Fibronectindetermination (Western blot method). The attached cells were suspendedin 1 ml of SDS sample buffer (cell-extract).

Western Blot: Spent culture medium (0.6 ml) was mixed with SDS samplebuffer (0.2 ml), and the cell-extract was boiled for 3 minutes beforeloading to SDS gel. Samples (80 ul/lane) were applied to a 6%-10% SDSgel and electrophoresis was conducted with 100 volts for 2 hours.Protein was transferred to a nitrocellulose membrane electrophoretically(30 min at 100 volts). The nitrocellulose blot was blocked with 5%non-fat dry milk in PBS (1-2 hours). The blot was then incubated withthe first antibodies (mouse anti-FN, specific to human FN, SIGMA F0916and mouse anti-beta actin, SIGMA A5316) and second antibody (Anti-mouseIgG AP conjugated, Promega S3721). The immuno-bands were developed withBCIP/NBT color development system (Promega S3771). Determination ofWestern band intensity: The band-images of Western blot were capturedwith a digital camera (3-5 pictures were taken per gel) and theintensity of bands was determined using “Image J” software.

FN concentrations were normalized with the cellular beta-Actinconcentrations. Fibronectin secreted into medium and inside Y-treatedcells were determined and compare to controls (DMSO-treated cells).

Results: This experiment shows that (1) there is a 46% reduction (54% ofcontrol) of FN secretion after Y-treatment and (2) the FN cellularcontent decrease 70% (30% of control) after the Y-treatment; (3) thereis no change of cellular beta-aecin concentration after the Y-treatment.

Experiment 36: Animal Study

-   -   Athymic Nu/Nu mice (2-3 months old) were transplanted sc with        ES2 (human ovarian cancer) cells.    -   Five days after the transplant (day one), mice were divided into        two groups (H and J) with two animals in each group.    -   Group H: On days 1-5, and 8-10 mice received daily drug        administration of Xanifolia-Y, by i.p. route at dose of 2.5        mg/kg.    -   Group J mice received no drug-treatment.        Result:

Group H: Mice received drug-treatment, tumor size is 10 mm in 10 days

Group J: Mice received no drug-treatment, tumor size is 18 mm in 10 days

The tumor size is 45% smaller in mice with drug than the mice with nodrug

-   -   in 10-day period.

Experiment 37: Aminal Study

Methods

-   -   Athymic Nu/Nu mice (5-6 weeks old) are divided into three groups        (0, P and Q) with 5-6 animals in each group.    -   On day 0, all mice were transplanted intra-peritoneally with ES2        (human ovarian cancer) cells.    -   Group 0: animals received no drug-treatment.    -   Group P: On days 4-8, 11-15, 18-22, 25-29, 32-36, 39-43, animals        received daily drug administration of Xanifolia-Y, by i.p. route        at dosage of 2.5 mg/kg    -   Group Q: On days 10-15, 18-22, 25-29, 32-36, 39-43, animals        received daily drug administration of Xanifolia-Y, by i.p. route        at dosage of 2.5 mg/kg.        Result:

The median survival time of tumor bearing mice without drug-treatment is24 days. The median survival time of tumor bearing mice withdrug-treatment starting on day 4 after tumor inoculation is 58 days(extension of life span of 141%); and The median survival time of tumorbearing mice with drug-treatment started on day 10 after tumorinoculation is 31 days (extension of life span of 29%).

Experiment 38: Inhibition of Cell Adhesion by Xanifolia-Y

Methods and Results: ES2 or Hey8A cells were plated in T25 flasks withmedium containing 5 ug/ml of Xanifolia-Y. Cultures were incubated for 5hours. Attached cells were removed from flasks by trypsinization and theamounts were counted. Compare to no drug controls, 86±4% of ES2 cellsand 67±8% of Hey8A cells were found attached to flasks under thiscondition. At 5 ug/ml Xanifolia-Y, over 90% of unattached cells arealive as determined by the trypan Blue exclusion assay and by theirability to re-attach to flasks when plating in medium withoutXanifolia-Y. However, with 10 ug/ml Xanifolia-Y, less than 40% of cellsattached to flasks and many of them are dead cells. This experimentshows that Xanifolia-Y inhibits cells adhesion process.

Experiment 39: Increase Synthesis of Angiopoietin-2 in ES2 Cells byXanifolia-Y

Methods: ES2 (human ovarian carcinoma cells) were grew in RPMI 1640medium. 4.5 million cells were seeded in a T75 flask and grown for 24hours before drug-treatment.

Drug-treatment: Cells cultures were treated with 5, 10 and 15 ug/ml(final concentration) of Xanifolia-Y3 [Y3-5, Y3-10, Y3-15]. or DMSOcontrol [D-10]. After 24 hours, cells were suspended in 1 ml of SDSsample buffer (cell-extract). Samples (80 ul/lane) were applied to a 10%SDS gel and electrophoresis was conducted with 100 volts for 2 hours.Protein was transferred to a nitrocellulose membraneelectrophoretically. The nitrocellulose blot was blocked with 5% non-fatdry milk in PBS. The blot was then incubated with the first antibodies(goat anti-Ang2, SIGMA A0851) and second antibody (donkey anti-goat APconjugated, Promega V115A). The immuno-bands were developed withBCIP/NBT color development system (Promega S3771).

Results: As shown in this Western Blot, a Angiopoietin-2 immuno-band wasobserved in extract from cells treated with 15 ug/ml Xanifolia-Y. No orminimal immuno-band of Angiopoietin-2 was observed in control and lowconcentration of xanifolia-Y. This result indicates that treatment ofXanifolia-Y in ES2 cells increase the cellular content (or synthesis) ofAngiopoietin-2. These results corroborate the results of Microarraystudies.

Experiment 40: Removal of the Sugar Moiety from Saponin by AcidHydrolysis

Method:

15 mg saponin was dissolved in 1 ml of Methanol. 1 ml of 2N HCl was thenadded. The mixture was refluxed in 80 C water bath for 5 hours. Thesolution was then neutralized by adding 2 ml of 1N NaOH (to final pH4-6). The aglycone was then extracted with ethylacetate 3 ml×2. Theextracts were collected and pooled. Further isolation of aglycone(sugar-removed saponin) was achieved by HPLC with isocratic elution of80-100% acetonitrile.

Experiment 41: Removal of the Acyl Group by Alkaline Hydrolysis

Method:

20 mg of saponin was dissolved in 0.5 ml of 1M NaOH. The solution wasincubated in 80 C water bath for 4 hours. It was cooled to roomtemperature before neutralized with 0.5 ml 1 N HCl (adjust pH to about3). The mixture was extracted with 2 ml 1-butanol 3 times. The butanolfractions were collected and lyophilized. The hydrolyzed saponin withfurther purified with HPLC in a C-18 column eluted with 25%acetonitrile.

Experiment 42: Analysis of Genesis of Blood Vessel in Xenograft TumorTreated with Compound Y

Method:

Athymic Nu/Nu mice (5-6 weeks old) are divided into two groups (1 and 2)with 5 animals in each group. On day 0, all mice were transplantedintra-peritoneally with one million ES2 (human ovarian cancer) cells.Animals were randomly divided into two groups:

Group 1: Control group. Animals did not receive drug-treatment.

Group 2: Drug-treatment group. On days 10-15 and 18-22, animals receiveddaily drug administration of Xanifolia-Y, by i.p. route at dosage of 2.5mg/kg.

Results: Animals showed high tumor burden after 18 days. Animals withhigh tumor burden were euthanized and solid tumors were taken out fromthese mice (between 18-27 days). Tumor tissues fixed with formalin atroom temperature. The fixed tissues were sectioned and stained withHaematoxylin and eosin (H&E). The red blood cells inside the micro bloodvessels were identified under a microscope. FIG. 44 shows that moreblood vessels were observed in the control Group 1 than those in thedrug-treated Group 2

Experiment 43: Determination of Cell Growth of Leishmania Parasites byMTT Assay

Method:

Leishmania parasites (Leishmania major: MRHO/SU/59/P/LV39) were grown inculture medium in a T75 flask at room temperature. Promastigotes ofLeishmania major (approximately 40 million per ml) were used in theexperiment. 1.2 ml cell culture was transferred to a well of the24-wells plate. Saponin Y10 (0.2 ml in medium) with differentconcentrations (final 6.25-200 ug/ml) was added to culture and cellswere grown for 1-5 days at room temperature. At the end ofdrug-treatment, 150 ul of MTT (5 mg/ml in PBS) was added to each welland incubated for 4 hours. Formazan formed in cells was dissolved withDMSO and the OD at 490 nm was determined by an ELISA reader.

Results:

this experiment shows that Y10 is cytotoxic to Leishmania Major(promastigotes) with IC50 approximately equal to 15 ug/ml. Experimentsare repeated with Y, ACH-Y, AKOH-Y, Mb5, ACH-Mb5, AKOH-Mb5 and Ba1.

Experiment 44: Adding the Acyl Group to Triterpene by Esterification

Method:

40 mg of triterpene core (fraction IV) was dissolved in 1 ml pyridine ina 50 ml tube. Reaction is started by adding 0.2 ml of acyl chloride(including Tigloyl chloride, angeloyl chloride or benzoyl chloride). Themixture is stirred for 3 days at room temperature. At the end ofreaction, 3 ml of NaHCO₃ is slowly added to the reaction mixture. Thesolution is then extracted 3 times with 10 ml of ethyl acetate which isthen evaporated under vacuum and at 45 C and lyophilization. The activeesterification products are purified with HPLC.

Experiment 45: Adding the Acyl Group to Triterpene by Esterification

Method:

40 mg of triterpene core (fraction IV) was dissolved in 1 ml pyridine ina 50 ml tube. Reaction is started by adding 0.2 ml of acyl chloride(including Tigloyl chloride, angeloyl chloride or benzoyl chloride). Themixture is stirred for 0.5 hr, 1 hr, 2 hrs, 3 hrs, 4 hrs, 8 hrs or 1 dayat room temperature. At the end of reaction, 3 ml of NaHCO₃ is slowlyadded to the reaction mixture. The solution is then extracted 3 timeswith 10 ml of ethyl acetate which is then evaporated under vacuum and at45 C and lyophilization. The active esterification products are purifiedwith HPLC.

What is claimed is:
 1. A composition for modulating the secretion orexpression of adhesion proteins or angiopoietins of cells, forinhibiting the metastasis of cancer cell in a subject, wherein thecomposition having a compound or its salt or acid or metabolite, isselected from the following:

wherein R1, R2 and R4 is selected from O-angeloyl, O-benzoyl, O-acetyl,O-alkanoyl, hydroxyl, CH2OH, COOH; R8, is OH; R3 is OH or H; R10, R11,R12, R13, R14, R15, R16 are CH4; R9 is CH2OH or CH3; wherein thecompound is selected from following a) A compound having structure(ACH-Z4):

b) A compound having structure (ACH-Y7):

c) A compound having structure (ACH-Y0):

d) A compound having structure (ACH-X):

e) A compound having structure (ACH-Mb5):

f) A compound having structure (ACH-Mb12):


2. The composition of claim 1, said compound having structure (ACH-Z4):


3. The composition of claim 1, said compound having structure (ACH-Y7):

or structure (ACH-Y0):


4. The composition of claim 1, said compound having structure (ACH-X):


5. The composition of claim 1, said compound having structure (ACH-Mb5):


6. The composition of claim 1, said compound having structure(ACH-Mb12):


7. The composition of claim 1, for inhibiting the metastasis of cancercell in a subject, wherein the compound modulates the expression of:ABL2, ADAMTS1, AKR1C3, AMIGO2, ANGPT2, ANKRD11, AP2B1, APEH, APLP2,ARL10C, ARMC4, ARMCX1, ARMCX6, ARNTL2, ATF3, ATP6V0E, ATP6V1B2,ATP6V1C1, ATP6V1C1, BCL2A1, BCL6, BRI3, BTD, C14orf109, C14orf78,C17orf32, C6orf65,C9orf10, C9orf103, CAD, CAV1, CAV2, CBLL1, CCL20,CD33L3, CEBPB, CEP4, CFH /// CFHL1, CHRDL1, CITED2, CLDN14, CLN8, CLTA,CNAP1, COG6, COL18A1, COL4A2, COL5A1, COL5A2, COL6A3, COPG, CPM, CPNE3,CPSF1, CSRP2BP, CSTB, CTNS, CXCL2, DDB1, DDIT3, DDX20, DKFZP564I1171,DKFZP586J0619, DUSP10, DUSP10, DYRK3, EEF2K, EFEMP1, EMP1, EVC, EVI2A,EXT2, FAM62A, FER1L3, FLJ14466, FLNA, FN1, GANAB, GDF15, GEM, GNPDA1,GPAA1, GPC6, GPNMB, GPNMB, GUSB, H2AFV, H2AFV, HDAC9, HDLBP, HECW2,HMGA2, HMOX1, HSDL2, HSPBAP1, HSPC196, HYOU1, IDS, IGFBP3, IKBKAP,INSIG1, IPO4, IRS2, JAG1, KDELR3, KIAA0251, KIAA0586, KIAA1211,KIAA1462, KIAA1706, KIAA1754, KRT18, KRT7, KRTAP4-7, LAMP2, LEPR,LEPREL1, LHFPL2, LIF, LOC286044, LOC339229, LOC90693, LRRC8E, MAFG,MAGED2, MCTP1, MGC16291, MGC19764, MGC5618, MRPS30, MRPS31, MTERFD3,MYH9, NAGA, NAV2, NCSTN, NEK9, NEU1, NFKBIZ, NMT2, NPC2, NSUN5C, NTNG1,NUP188, OACT2, 0S9, P4HA1, P8, PALM2-AKAP2, PALM2-AKAP2, PARVA, PBX2,PDE4DIP, PDIA4, PDIA6, PEG10, PHF19, PIK4CA, PLEKHM1, PLOD1, PLOD2,PPP1R15A, PPP1R15A, PRKDC, PRSS23, PRSS23, PSEN2, PSMD1, PTPRF, PTPRJ,RAB32, RAB9A, RG9MTD1, RGS4, RHOQ, RND3, RNF25, RNPEP /// UBE2V1 /// Kua/// Kua-UEV, RNU17D, ROBO4, RRAGC, RRS1, SEC31L1, SERPINB2, SERPINB7,SESN2, SGEF, SGSH, SKIV2L, SLC25A21, SLC35A3, SLC3A2, SMARCA1, SNAPC1,SNF1LK, SPOCD1, SPTAN1, SQSTM1, ST3GAL6, STC2, STX3A, TFPI2, TGFBI,TGM2, THRAP1, TLN1, TMEM60, TNFAIP3, TRIB3, TRIO, TSC2, UAP1L1, UBAP2L,UPP1, URB, USP11, USP5, VDR, WDR4, YTHDF2, ZCCHC9, ZDHHC20, ZFHX1B,ZNF185, ZNF278, ZNF690, ZNF697, ITGAV, LAMA4, LAMB2, LAMC1, LAMB1,LAMA5, LAMC1, LAMA2, LAMB1, LAMA5, SCAMP1, TICAM2, CAMK2B, DL1, ICAM3,CEECAM1, ICAM5, CAMK1G, CAMSAP1, MCAM, CAMTA1, CKN1, ALCAM, DCAMKL2,CEACAM3, CAMK2D, CAMK2B, SCAMP5, CAMK4, NCAM1, CAMK2G, MYH10, MYO1D,MYO5A, MYO1C, MYLK, MYO6, MYLC2PL, MYO10, BECN1, MYO1E, TPM3, M-RIP,MYO1B, MYL6, MYOHD1, BECN1, TPM4, MYLK, MYOHD1, LOC221875, LOC402643,MYO15B, LOC129285, MYH11, MYO9B, CTNNAL1, CDH13, CDH12, CTNNB1, CDH5,CTNND1, CDH2, PCDHB16, CTNNA1, CELSR2, PCDHB6, PCDHB7, CTNND2, PCDHGC3,PCDHGB4, PCDHGA8, PCDHGA12, PCDHGC5, PCDHGC4, PCDHGB7, PCDHGB6, PCDHGB5,PCDHGB3, PCDHGB2, PCDHGB1, PCDHGA11, PCDHGA10, PCDHGA9, PCDHGA7,PCDHGA6, PCDHGA5, PCDHGA4, PCDHGA3, PCDHGA2, PCDHGA1, CTNND1, CDH23,PCDHB12, PCDHB10, PCDH18, CDH20, PCDH9, PCDHGA12, PCDHGA11, PCDHGA10,PCDHGA6, PCDHGA5, PCDHGA3, PCDH7, CDH18, CDH6, CCBE1, COL10A1, COL12A1,COL13A1, COL1A1, COL21A1, COL4A1, COL4A5, COL4A6, COL6A1, COL6A2,COL9A1, MMP9, P4HA1, P4HA2, P4HB, PCOLCE, PCOLCE2, PCOTH, PLODS, CIB1,ILK, ITGA2, ITGA3, ITGA4, ITGA6, ITGAV, ITGB1, ITGB1BP1, ITGB2, ITGB5,ITGBL1, TNC, EMILIN1, ICAM1, HSPG2, HPSE, HS2ST1, or SDC2.
 8. Thecomposition of claim 1, said compound having structure (ACH-Y0):


9. A method for modulating the secretion or expression of adhesionproteins of cells, or for inhibiting the metastasis of cancer cells, ina subject, comprising contacting said subject with an effective amountof a compound or its salt, acid thereof selected from the following: a)A compound having structure (ACH-Z4):

b) A compound having structure (ACH-Y10):

c) A compound having structure (ACH-Y8):

d) A compound having structure (ACH-Y7):

e) A compound having structure (ACH-Y0):

f) A compound having structure (ACH-E):

g) A compound having structure (ACH-X):

h) A compound having structure (ACH-Mb5):

i) A compound having structure (ACH-Mb12):


10. The method of claim 9, wherein the compound has structure (ACH-Z4):


11. The method of claim 9, wherein the compound has structure (ACH-Y10):


12. The method of claim 9, wherein the compound has structure (ACH-Y8):


13. The method of claim 9, wherein the compound has structure (ACH-Y7):


14. The method of claim 9, wherein the compound has structure (ACH-Y0):


15. The method of claim 9, wherein the compound has structure (ACH-E):


16. The method of claim 9, wherein the compound has structure (ACH-X):


17. The method of claim 9, wherein the compound has structure (ACH-Mb5):


18. The method of claim 9, wherein the compound has structure(ACH-Mb12):


19. A composition for treating parasites of leishmaniases, amoebiasis,trypanosomiasis, toxoplasmosis or malaria in a subject, wherein thecomposition has an effective amount of compound, or its salt, acid orester thereof, selected from the following: a) A compound havingstructure ((ACH-Y)):

b) A compound having structure (ACH-Z4):

c) A compound having structure (ACH-Y7):

d) A compound having structure (ACH-Y0):

e) A compound having structure (ACH-X):


20. The composition of claim 19, said compound having structure (ACH-Y):